Antigen-binding molecule inducing immune response to target antigen

ABSTRACT

The present inventors have discovered that in living organisms that have received an antigen-binding molecule containing an antigen-binding domain whose binding activity to an antigen changes depending on ion concentration conditions and containing an FcRn-binding domain having FcRn-binding activity in a neutral pH range, immune responses to the antigen are induced. Furthermore, the present inventors have discovered that in living organisms that have received an antigen-binding molecule containing an antigen-binding domain whose binding activity to an antigen changes depending on ion concentration conditions and containing an FcRn-binding domain having FcRn-binding activity in a neutral pH range, immune responses to the antigen are induced, and also the antigen-binding molecule has cytotoxicity or antiproliferative action against cancer cells, foreign biological species, or such that express the antigen to which the antigen-binding molecule binds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/347,448, filed on Mar. 26, 2014, which is the National Stage ofInternational Application Serial No. PCT/JP2012/075043, filed on Sep.28, 2012, which claims the benefit of Japanese Application Serial No.2011-216958, filed on Sep. 30, 2011.

TECHNICAL FIELD

The present invention provides pharmaceutical compositions comprising asan active ingredient an antigen-binding molecule that induces an immuneresponse to a target antigen, or therapeutic methods that use thepharmaceutical compositions. The present invention also providespharmaceutical compositions comprising as an active ingredient, anantigen-binding molecule that induces the above-mentioned immuneresponse and also has cytotoxicity (cytotoxic activity) orantiproliferative action (cell proliferation inhibitory activity)against cells expressing a target antigen, or therapeutic methods thatuse the pharmaceutical compositions.

BACKGROUND ART

To date, attempts have been made to develop a number of therapeuticvaccines directed to tumor cells. This is because it is considered thatthere are qualitative or quantitative differences between tumor cellsand normal cells that may be recognized by the immune system of a livingorganism, and the immune system stimulated by active and specificsensitization by vaccines utilizing such differences (neoepitopes) canrecognize and eliminate tumor cells.

To bring about such anti-tumor response, at least two conditions mayhave to be met. Firstly, the tumor cells must express an antigen thatdoes not appear in normal cells, or express an antigen to such an extentthat normal cells and tumor cells can be distinguished solely in aqualitative manner. Secondly, the immune system must be activated byvaccines or such in order to react with the antigen of interest. A majorobstacle in tumor immunotherapy is considered to be that theimmunogenicity of tumors is particularly weak in humans.

In recent years, tumor-related and tumor-specific antigens includingsuch neoepitopes that may constitute targets to be attacked by theimmune system have been discovered. Nonetheless, the immune systemcannot eliminate tumors expressing such neoepitopes, and this may be dueto insufficient immune response to these neoepitopes, rather than due tothe absence of neoepitopes.

Two general strategies have been developed for the purpose of cell-basedcancer immunotherapies. One of them is adoptive immunotherapy wheretumor-reactive T lymphocytes expanded in vitro are reintroduced into apatient, and the other is active immunotherapy which uses tumor cells toinduce systemic tumor response by triggering new or stronger immuneresponse to a tumor antigen.

Tumor vaccines based on active immunotherapy have been prepared byvarious methods. To induce immune response to a tumor antigen,irradiated tumor cells mixed with an immune-stimulating adjuvant such asBacillus Calmette Guerin (BCG) (Non-Patent Document 1), tumor cellsgenetically modified to produce, for example, cytokines (Non-PatentDocument 2), and alienated autologous tumor cells (Non-Patent Document3) have been prepared. However, the immunogenicity of the tumor cells islow, and this is considered to be due to the quantity of the tumorantigen, not the quality.

On the other hand, antibodies are known to induce humoral immuneresponses (production of antibodies against an antigen) and cellularimmune responses (production of CD8-positive T cells against an antigen)to antigens by cross-presenting bound antigens to antigen-presentingcells, and it has been reported that administration of an antibody caninduce acquired immunity to an antigen (Non-Patent Document 4).Recently, for the anti-tumor effect by an anti-HER2 antibody, it hasbeen shown in an in vivo mouse model that acquired immunity to the HER2antigen induced by administration of the antibody plays a more importantrole than the direct ADCC of the administered antibody (Non-PatentDocument 5). In fact, in clinical use of Herceptin, which is an IgG1subclass antibody drug against HER2, acquired immunity was induced byHerceptin administration, and humoral immune response to HER2 wasobserved (Non-Patent Document 6). Since patients in whom Herceptinadministration was effective particularly showed an increased anti-HER2antibody titer, induction of acquired immunity by Herceptinadministration was considered to play an important role in theanti-tumor effect.

Antibodies are highly stable in blood and have few side effects, and aretherefore drawing attention as pharmaceuticals (Non-Patent Documents 7and 8). Many studies have been carried out so far on antibody-dependentcellular cytotoxicity (hereinafter denoted as ADCC) andcomplement-dependent cytotoxicity (hereinafter denoted as CDC), whichare effector functions of IgG class antibodies. It has been reportedthat in the human IgG class, antibodies of the IgG1 subclass have thehighest ADCC activity and CDC activity (Non-Patent Document 9).Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), whichis phagocytosis of target cells mediated by IgG class antibodies, isalso suggested to be one of the antibody effector functions (Non-PatentDocuments 10 and 11). Since IgG1 subclass antibodies can exert theseeffector functions against tumors, IgG1 subclass antibodies are used formost antibody pharmaceuticals against cancer antigens.

In order for IgG antibodies to mediate ADCC and ADCP activities, the Fcregion of the IgG antibodies must bind to antibody receptors(hereinafter denoted as FcγR) that are present on the surface ofeffector cells such as killer cells, natural killer cells, and activatedmacrophages. In humans, isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, andFcγRIIIb have been reported as members of the FcγR protein family, andtheir respective allotypes have been reported as well (Non-PatentDocument 12).

Enhancement of cytotoxic effector functions such as ADCC and ADCP hasbeen drawing attention as a promising means for enhancing the antitumoreffects of anticancer antibodies. Importance of FcγR-mediated effectorfunctions aimed for antitumor effects of antibodies has been reportedusing mouse models (Non-Patent Documents 13 and 14). Furthermore, it wasobserved that clinical effects in humans correlated with thehigh-affinity polymorphic allotype (V158) and the low-affinitypolymorphic allotype (F158) of FcγRIIIa (Non-Patent Document 15). Thesereports suggest that antibodies with an Fc region optimized for bindingto a specific FcγR mediates stronger effector functions, and therebyexert more effective antitumor effects. The balance between the affinityof antibodies against the activating receptors including FcγRIa,FcγRIIa, FcγRIIIa, and FcγRIIIb, and the inhibitory receptors includingFcγRIIb is an important factor in optimizing antibody effectorfunctions. Enhancing the affinity to activating receptors may giveantibodies a property to mediate stronger effector functions (Non-PatentDocument 16), and therefore has been reported in various reports to dateas an antibody engineering technique for improving or enhancing theantitumor activity of antibody pharmaceuticals against cancer antigens.

Regarding binding between the Fc region and FcγR, several amino acidresidues in the antibody hinge region and the CH2 domain, and a sugarchain added to Asn at position 297 (EU numbering) bound to the CH2domain have been shown as being important (Non-Patent Documents 9, 17,and 18). Focusing on this binding site, studies have so far been carriedout on mutants of the Fc region having various FcγR binding properties,and Fc region mutants with higher affinity to activating FcγR have beenobtained (Patent Documents 1 and 2). For example, Lazar et al. havesucceeded in increasing the binding of human IgG1 to human FcγRIIIa(V158) by approximately 370 fold by substituting Ser at position 239,Ala at position 330, and Ile at position 332 (EU numbering) of humanIgG1 with Asn, Leu, and Glu, respectively (Non-Patent Document 19 andPatent Document 2). The ratio of binding to FcγRIIIa and FcγRIIb (A/Iratio) for this mutant was approximately 9-fold that of the wild type.Furthermore, Shinkawa et al. have succeeded in increasing the binding toFcγRIIIa up to approximately 100 fold by removing fucose from the sugarchain added to Asn at position 297 (EU numbering) (Non-Patent Document20). These methods can greatly improve the ADCC activity of human IgG1compared to that of naturally-occurring human IgG1.

While there are many reports, as described above, on methods forenhancing ADCC by antibody engineering, no reports have been made todate on antibody engineering techniques for enhancing or improvinginduction of acquired immunity by antibody administration. There is areport on methods for inducing acquired immunity against a cancerantigen, in which a cancer antigen against which acquired immunity isdesired to be induced is fused with an antibody that binds to ahigh-mannose receptor or DEC-205 expressed on antigen presenting cells,thereby promoting incorporation and presentation of the cancer antigenby antigen presenting cells (Non-Patent Document 21). However, in thesemethods the target of antibody binding is not a cancer antigen as in thecase of the above-mentioned anti-HER2 antibody. That is, since thesemethods induce acquired immunity against a cancer antigen fused to theantibody itself, the antibody itself cannot bind to the cancer antigen,and has the disadvantage of not being able to exhibit direct action onthe cancer antigen. Furthermore, since this method induces acquiredimmunity not only against the cancer antigen fused to the antibody butalso against the antibody itself used for targeting antigen-presentingcells, anti-drug antibodies will emerge and this leads to weakening ofthe effects. Therefore, this method may not be preferable fortherapeutic purposes.

According to the above, while it is desirable to induce acquiredimmunity to a target antigen by administering an antigen-bindingmolecule having binding activity to the target antigen, there has beenno reports on engineering techniques for improving or enhancing acquiredimmunity by such methods.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] WO2000/042072-   [Patent Document 2] WO2006/019447

Non-Patent Documents

-   [Non-patent Document 1] Oettgen, H. F., and Old, L. J., The history    of cancer immunotherapy, Biological Therapy of Cancer (1991) 87-119    DeVita et al. ed.-   [Non-patent Document 2] Zatloukal K, Schmidt W, Cotten M, Wagner E,    Stingl G, Birnstiel M L., Somatic gene therapy for cancer: the    utility of transferrinfection in generating ‘tumor vaccines’,    Gene (1993) 135, 199-207-   [Non-patent Document 3] Bronte V, Tsung K, Rao J B, Chen P W, Wang    M, Rosenberg S A, Restifo N P., IL-2 enhances the function of    recombinant poxvirus-based vaccines in the treatment of established    pulmonary metastases, J. Immunol. (1995) 154, 5282-5292-   [Non-patent Document 4] Adams G P, Weiner L M., Monoclonal antibody    therapy of cancer, Nat. Biotechnol. (2005) 23, 1147-1157-   [Non-patent Document 5] Park S, Jiang Z, Mortenson E D, Deng L,    Radkevich-Brown O, Yang X, Sattar H, Wang Y, Brown N K, Greene M,    Liu Y, Tang J, Wang S, Fu Y X., The therapeutic effect of    anti-HER2/neu antibody depends on both innate and adaptive immunity,    Cancer Cell (2010) 18, 160-170 (2010)-   [Non-patent Document 6] Taylor C, Hershman D, Shah N, Suciu-Foca N,    Petrylak D P, Taub R, Vandat L, Cheng B, Pegram M, Knutson K L,    Clynes R. Augmented HER-2 specific immunity during treatment with    trastuzumab and chemotherapy, Clin. Cancer Res, (2007) 13, 5133-43-   [Non-patent Document 7] anice M Reichert, Clark J Rosensweig, Laura    B Faden & Matthew C Dewitz, Monoclonal antibody successes in the    clinic, Nat. Biotechnol. (2005) 23, 1073-1078-   [Non-patent Document 8] avlou AK, Belsey M J., The therapeutic    antibodies market to 2008, Eur. J. Pharm. Biopharm. (2005) 59(3),    389-396-   [Non-patent Document 9] Clark, M., Antibody Engineering IgG Effector    Mechanisms, Chemical Immunology (1997), 65, 88-110-   [Non-patent Document 10] Horton H M, Bernett M J, Pong E, Peipp M,    Karki S, Chu S Y, Richards J O, Vostiar I, Joyce P F, Repp R,    Desjarlais J R, Zhukovsky E A., Potent in vitro and in vivo activity    of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma    and leukemia, Cancer Res. (2008) 68, 8049-8057-   [Non-patent Document 11] Zalevsky J, Leung I W, Karki S, Chu S Y,    Zhukovsky E A, Desjarlais J R, Carmichael D F, Lawrence C E., The    impact of Fc engineering on an anti-CD19 antibody: increased Fcγ    receptor affinity enhances B-cell clearing in nonhuman primates,    Blood (2009) 113, 3735-3743-   [Non-patent Document 12] Jefferis R, Lund J., Interaction sites on    human IgG-Fc for FcgammaR: current models, Immunol. Lett. (2002) 82,    57-65-   [Non-patent Document 13] Clynes, R., Yoshizumi, T., Moroi, Y,    Houghton, A. N., and Ravetch, J. V., Fc Receptors are required for    passive and active immunity to melanoma, Proc. Natl. Acad. Sci.    U.S.A (1998) 95, 652-656-   [Non-patent Document 14] Clynes R A, Towers T L, Presta L C; Ravetch    J V., Inhibitory Fc receptors modulate in vivo cytoxicity against    tumor targets, Nat. Med. (2000) 6, 443-446-   [Non-patent Document 15] Cartron G, Dacheux L, Salles G,    Solal-Celigny P, Bardos P, Colombat P, Watier H., Therapeutic    activity of humanized anti-CD20 monoclonal antibody and polymorphism    in IgG Fc receptor FcgammaRIIIa gene, Blood (2002) 99, 754-758-   [Non-patent Document 16] Nimmerjahn F, Ravetch J V., Divergent    immunoglobulin g subclass activity through selective Fc receptor    binding, Science (2005), 310, 1510-1512-   [Non-patent Document 17] Greenwood J, Clark M, Waldmann H.,    Structural motifs involved in human IgG antibody effector functions,    Eur. J. Immunol. 23, 1098-1104 (1993)-   [Non-patent Document 18] Morgan A, Jones N D, Nesbitt A M, Chaplin    L, Bodmer M W, Emtage J S., The N-terminal end of the CH2 domain of    chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI    and Fc gamma RIII binding, Immunology (1995) 86, 319-324-   [Non-patent Document 19] Lazar G A, Dang W, Karki S, Vafa O, Peng J    S, Hyun L, Chan C, Chung H S, Eivazi A, Yoder S C, Vielmetter J,    Carmichael D F, Hayes R J, Dahiyat B I., Engineered antibody Fc    variants with enhanced effector function, Proc. Natl. Acad. Sci.    U.S.A (2006) 103, 4005-4010-   [Non-patent Document 20] Shinkawa T, Nakamura K, Yamane N,    Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M,    Yamasaki M, Hanai N, Shitara K., The absence of fucose but not the    presence of galactose or bisecting N-acetylglucosamine of human IgG1    complex-type oligosaccharides shows the critical role of enhancing    antibody-dependent cellular cytotoxicity, J. Biol. Chem. (2003) 278,    3466-3473-   [Non-patent Document 21] Tsuji T, Matsuzaki J, Kelly M P,    Ramakrishna V, Vitale L, He L Z, Keler T, Odunsi K, Old L J, Ritter    G, Gnjatic S., Antibody-Targeted NY-ESO-1 to Mannose Receptor or    DEC-205 In Vitro Elicits Dual Human CD8+ and CD4+ T Cell Responses    with Broad Antigen Specificity, J. Immunol. (2011) 186, 1218-1827

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide pharmaceuticalcompositions comprising as an active ingredient an antigen-bindingmolecule that induces an immune response in subjects affected withcancer or infected with foreign biological species when administered tothese subjects, or therapeutic methods that use the pharmaceuticalcompositions. Another objective is to provide pharmaceuticalcompositions comprising as an active ingredient an antigen-bindingmolecule that induces the above-mentioned immune response and also hascytotoxicity (cytotoxic activity) or antiproliferative action (cellproliferation inhibitory activity) against cancer cells or infectingforeign biological species, or therapeutic methods that use thepharmaceutical compositions.

Means for Solving the Problems

The present inventors have discovered that in living organisms that havereceived an antigen-binding molecule containing an antigen-bindingdomain whose binding activity against an antigen changes depending onion concentration conditions and containing an FcRn-binding domainhaving FcRn-binding activity under a neutral pH range, immune responsesto the antigen are induced. Furthermore, the present inventors havediscovered that in living organisms that have received anantigen-binding molecule containing an antigen-binding domain whoseantigen-binding activity changes depending on ion concentrationconditions and an FcRn-binding domain having FcRn-binding activity undera neutral pH range, immune responses to the antigen are induced, and theantigen-binding molecule can also have cytotoxicity or antiproliferativeeffect against cancer cells, foreign biological species, or the likethat express the antigen to which the antigen-binding molecule binds.Based on these findings, the present inventors have elucidated that theantigen-binding molecules of the present invention are useful aspharmaceutical compositions for inducing an immune response in a subjectinfected with a foreign biological species or affected with cancer whenadministered to the subject. The present inventors have also elucidatedthat the antigen-binding molecules of the present invention are usefulas pharmaceutical compositions that, when administered to a subjectinfected with a foreign biological species or affected with cancer,induce an immune response in the subject and also have cytotoxicity orantiproliferative effect against the cancer cells and foreign biologicalspecies. Methods for producing these pharmaceutical compositions havealso been discovered.

More specifically, the present invention provides [1] to [47] below:

[1] a pharmaceutical composition that induces an immune response to anantigen, which comprises as an active ingredient an antigen-bindingmolecule, wherein the antigen-binding molecule comprises anantigen-binding domain whose binding activity to the antigen changesdepending on an ion concentration condition and comprises anFcRn-binding domain having binding activity to FcRn in a neutral pHrange;[2] the pharmaceutical composition of [1], wherein the ion concentrationis a calcium ion concentration;[3] the pharmaceutical composition of [2], wherein the antigen-bindingdomain is an antigen-binding domain whose antigen-binding activity ishigher under a high calcium ion concentration condition than under a lowcalcium ion concentration condition;[4] the pharmaceutical composition of [1], wherein the ion concentrationcondition is a pH condition;[5] the pharmaceutical composition of [4], wherein the antigen-bindingdomain is an antigen-binding domain whose antigen-binding activity ishigher in a neutral pH range than under an acidic pH range;[6] the pharmaceutical composition of any one of [1] to [5], wherein theantigen-binding molecule has neutralizing activity against the antigen;[7] the pharmaceutical composition of any one of [1] to [6], wherein theantigen-binding molecule has cytotoxic activity against a cellexpressing the antigen;[8] the pharmaceutical composition of any one of [1] to [7], wherein theFcRn-binding domain comprises an antibody Fc region;[9] the pharmaceutical composition of [8], wherein the Fc region is anFc region in which at least one or more amino acids selected from thegroup consisting of amino acids at positions 257, 308, 428, and 434according to EU numbering in the Fc region are different from aminoacids at corresponding positions in a naturally-occurring Fc region;[10] the pharmaceutical composition of [8] or [9], wherein the Fc regioncomprises at least one or more amino acids selected from the groupconsisting of:Ala at amino acid position 257;Pro at amino acid position 308;Leu at amino acid position 428; andTyr at amino acid position 434,according to EU numbering in the Fc region;[11] the pharmaceutical composition of any one of [8] to [10], whereinthe Fcγ receptor-binding activity of the Fc region is higher than thatof a naturally-occurring human IgG Fc region in which the sugar chainattached at position 297 according to EU numbering is afucose-containing sugar chain;[12] the pharmaceutical composition of [11], wherein the Fcγ receptor isFcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F);[13] the pharmaceutical composition of [11] or [12], wherein the Fcregion is an Fc region in which at least one or more amino acidsselected from the group consisting of amino acids at positions 221, 222,223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256,258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379,380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 accordingto EU numbering in the Fc region are different from amino acids atcorresponding positions in a naturally-occurring Fc region;[14] the pharmaceutical composition of any one of [11] to [13], whereinthe Fc region comprises at least one or more amino acids selected fromthe group consisting of:either Lys or Tyr at amino acid position 221;any one of Phe, Trp, Glu, and Tyr at amino acid position 222;any one of Phe, Trp, Glu, and Lys at amino acid position 223;any one of Phe, Trp, Glu, and Tyr at amino acid position 224;any one of Glu, Lys, and Trp at amino acid position 225;any one of Glu, Gly, Lys, and Tyr at amino acid position 227;any one of Glu, Gly, Lys, and Tyr at amino acid position 228;any one of Ala, Glu, Gly, and Tyr at amino acid position 230;any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 231;any one of Glu, Gly, Lys, and Tyr at amino acid position 232;any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 233;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;

any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and

Tyr at amino acid position 236;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 237;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 238;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Thr, Val, Trp, and Tyr at amino acid position 239;any one of Ala, Ile, Met, and Thr at amino acid position 240;any one of Asp, Glu, Leu, Arg, Trp, and Tyr at amino acid position 241;any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at amino acid position243;His at amino acid position 244;Ala at amino acid position 245;any one of Asp, Glu, His, and Tyr at amino acid position 246;any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at aminoacid position 247;any one of Glu, His, Gln, and Tyr at amino acid position 249;either Glu or Gln at amino acid position 250;Phe at amino acid position 251;any one of Phe, Met, and Tyr at amino acid position 254;any one of Glu, Leu, and Tyr at amino acid position 255;any one of Ala, Met, and Pro at amino acid position 256;any one of Asp, Glu, His, Ser, and Tyr at amino acid position 258;any one of Asp, Glu, His, and Tyr at amino acid position 260;any one of Ala, Glu, Phe, Ile, and Thr at amino acid position 262;any one of Ala, Ile, Met, and Thr at amino acid position 263;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Trp, and Tyr at amino acid position 264;any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Val, Trp, and Tyr at amino acid position 265;any one of Ala, Ile, Met, and Thr at amino acid position 266;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Thr, Val, Trp, and Tyr at amino acid position 267;any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr,Val, and Trp at amino acid position 268;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 269;any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr,Trp, and Tyr at amino acid position 270;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 271;any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 272;either Phe or Ile at amino acid position 273;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 274;either Leu or Trp at amino acid position 275;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 276;any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, and Trp at amino acid position 278;Ala at amino acid position 279;any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at amino acidposition 280;any one of Asp, Lys, Pro, and Tyr at amino acid position 281;any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 282;any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at aminoacid position 283;any one of Asp, Glu, Leu, Asn, Thr, and Tyr at amino acid position 284;any one of Asp, Glu, Lys, Gln, Trp, and Tyr at amino acid position 285;any one of Glu, Gly, Pro, and Tyr at amino acid position 286;any one of Asn, Asp, Glu, and Tyr at amino acid position 288;any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at amino acidposition 290;any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at amino acid position291;any one of Ala, Asp, Glu, Pro, Thr, and Tyr at amino acid position 292;any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, and Tyr at amino acid position 293;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 294;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 295;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, and Val at amino acid position 296;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 297;any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr,Val, Trp, and Tyr at amino acid position 298;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Val, Trp, and Tyr at amino acid position 299;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, and Trp at amino acid position 300;any one of Asp, Glu, His, and Tyr at amino acid position 301;Ile at amino acid position 302;any one of Asp, Gly, and Tyr at amino acid position 303;any one of Asp, His, Leu, Asn, and Thr at amino acid position 304;any one of Glu, Ile, Thr, and Tyr at amino acid position 305;any one of Ala, Asp, Asn, Thr, Val, and Tyr at amino acid position 311;Phe at amino acid position 313;Leu at amino acid position 315;either Glu or Gln at amino acid position 317;any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at amino acidposition 318;any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp,and Tyr at amino acid position 320;any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, andTyr at amino acid position 322;Ile at amino acid position 323;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp,and Tyr at amino acid position 324;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr,Val, Trp, and Tyr at amino acid position 326;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Arg, Thr, Val, Trp, and Tyr at amino acid position 327;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 329;any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 330;any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, andTyr at amino acid position 331;any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr,Val, and Tyr at amino acid position 333;any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at amino acid position334;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val,Trp, and Tyr at amino acid position 335;any one of Glu, Lys, and Tyr at amino acid position 336;any one of Glu, His, and Asn at amino acid position 337;any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr atamino acid position 339;either Ala or Val at amino acid position 376;either Gly or Lys at amino acid position 377;Asp at amino acid position 378;Asn at amino acid position 379;any one of Ala, Asn, and Ser at amino acid position 380;either Ala or Ile at amino acid position 382;Glu at amino acid position 385;Thr at amino acid position 392;Leu at amino acid position 396;Lys at amino acid position 421;Asn at amino acid position 427;either Phe or Leu at amino acid position 428;Met at amino acid position 429;Trp at amino acid position 434;Ile at amino acid position 436; andany one of Gly, His, Ile, Leu, and Tyr at amino acid position 440;according to EU numbering in the Fc region;[15] the pharmaceutical composition of any one of [11] to [14], whereinthe naturally-occurring Fc region is an Fc region of any one of humanIgG1, human IgG2, human IgG3, and human IgG4 in which the sugar chainattached at position 297 according to EU numbering is afucose-containing sugar chain;[16] the pharmaceutical composition of any one of [11] to [15], whereinthe Fc region is modified so that the percentage of the Fc region towhich a fucose-deficient sugar chain is attached, or bisectingN-acetylglucosamine is added, at position 297 according to EU numberingin the Fc region, will become higher.[17] a method for inducing an immune response in a living organism,which comprises the step of administering the antigen-binding moleculeof any one of [1] to [16] to the living organism;[18] a method for producing an antigen-binding molecule that induces animmune response, which comprises imparting FcRn-binding activity in aneutral pH range to an FcRn-binding domain that is contained in anantigen-binding molecule containing an antigen-binding domain whoseantigen-binding activity changes depending on an ion concentrationcondition;[19] the method of [18], wherein the ion concentration is a calcium ionconcentration;[20] the method of [19], wherein the antigen-binding domain is anantigen-binding domain whose antigen-binding activity is higher under ahigh calcium ion concentration condition than under a low calcium ionconcentration condition;[21] the method of [18], wherein the ion concentration condition is a pHcondition;[22] the method of [21], wherein the antigen-binding domain is anantigen-binding domain whose antigen-binding activity is higher in aneutral pH range than in an acidic pH range;[23] the method of any one of [18] to [22], wherein the antigen-bindingmolecule has neutralizing activity against the antigen;[24] the method of any one of [18] to [23], wherein the antigen-bindingmolecule has cytotoxic activity against a cell expressing the antigen;[25] the method of any one of [18] to [24], wherein the FcRn-bindingdomain comprises an antibody Fc region;[26] the method of [25], comprising the step of substituting at leastone or more amino acids selected from the group consisting of aminoacids at positions 239, 252, 257, 286, 307, 308, 428, and 434 accordingto EU numbering in the Fc region;[27] the method of [25] or [26], comprising the step of performing atleast one or more amino acid substitutions selected from the groupconsisting of:amino acid substitution with Ala at position 257;amino acid substitution with Pro at position 308;amino acid substitution with Leu at position 428; andamino acid substitution with Tyr at position 434,according to EU numbering in the Fc region;[28] the method of any one of [25] to [27], comprising the step ofenhancing the Fcγ receptor-binding activity of the Fc region as comparedto that of a naturally-occurring human IgG Fc region in which the sugarchain attached at position 297 according to EU numbering is afucose-containing sugar chain;[29] the method of [28], wherein the Fcγ receptor is FcγRIa, FcγRIIa(R),FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F);[30] the method of [28] or [29], comprising the step of substituting atleast one or more amino acids selected from the group consisting ofamino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246,247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281,282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427,428, 429, 434, 436, and 440 according to EU numbering in the Fc region;[31] the method of any one of [28] to [30], comprising the step ofperforming at least one or more amino acid substitutions selected fromthe group consisting of:amino acid substitution with either Lys or Tyr at position 221;amino acid substitution with any one of Phe, Trp, Glu, and Tyr atposition 222;amino acid substitution with any one of Phe, Trp, Glu, and Lys atposition 223;amino acid substitution with any one of Phe, Trp, Glu, and Tyr atposition 224;amino acid substitution with any one of Glu, Lys, and Trp at position225;amino acid substitution with any one of Glu, Gly, Lys, and Tyr atposition 227;amino acid substitution with any one of Glu, Gly, Lys, and Tyr atposition 228;amino acid substitution with any one of Ala, Glu, Gly, and Tyr atposition 230;amino acid substitution with any one of Glu, Gly, Lys, Pro, and Tyr atposition 231;amino acid substitution with any one of Glu, Gly, Lys, and Tyr atposition 232;amino acid substitution with any one of Ala, Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at position 233;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 234;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 235;amino acid substitution with any one of Ala, Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 236;amino acid substitution with any one of Asp, Glu, Phe, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position237;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position238;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position239;amino acid substitution with any one of Ala, Ile, Met, and Thr atposition 240;amino acid substitution with any one of Asp, Glu, Leu, Arg, Trp, and Tyrat position 241;amino acid substitution with any one of Leu, Glu, Leu, Gln, Arg, Trp,and Tyr at position 243;amino acid substitution with His at position 244;amino acid substitution with Ala at position 245;amino acid substitution with any one of Asp, Glu, His, and Tyr atposition 246;amino acid substitution with any one of Ala, Phe, Gly, His, Ile, Leu,Met, Thr, Val, and Tyr at position 247;amino acid substitution with any one of Glu, His, Gln, and Tyr atposition 249;amino acid substitution with either Glu or Gln at position 250;amino acid substitution with Phe at position 251;amino acid substitution with any one of Phe, Met, and Tyr at position254;amino acid substitution with any one of Glu, Leu, and Tyr at position255;amino acid substitution with any one of Ala, Met, and Pro at position256;amino acid substitution with any one of Asp, Glu, His, Ser, and Tyr atposition 258;amino acid substitution with any one of Asp, Glu, His, and Tyr atposition 260;amino acid substitution with any one of Ala, Glu, Phe, Ile, and Thr atposition 262;amino acid substitution with any one of Ala, Ile, Met, and Thr atposition 263;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position264;amino acid substitution with any one of Ala, Leu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyr atposition 265;amino acid substitution with any one of Ala, Ile, Met, and Thr atposition 266;amino acid substitution with any one of Asp, Glu, Phe, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at position 267;amino acid substitution with any one of Asp, Glu, Phe, Gly, Ile, Lys,Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at position 268;amino acid substitution with any one of Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at position 269;amino acid substitution with any one of Glu, Phe, Gly, His, Ile, Leu,Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position 270;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 271;amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Lys,Leu, Met, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at position 272;amino acid substitution with either Phe or Ile at position 273;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 274;amino acid substitution with either Leu or Trp at position 275;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 276;amino acid substitution with any one of Asp, Glu, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 278;amino acid substitution with Ala at position 279;amino acid substitution with any one of Ala, Gly, His, Lys, Leu, Pro,Gln, Trp, and Tyr at position 280;amino acid substitution with any one of Asp, Lys, Pro, and Tyr atposition 281;amino acid substitution with any one of Glu, Gly, Lys, Pro, and Tyr atposition 282;amino acid substitution with any one of Ala, Gly, His, Ile, Lys, Leu,Met, Pro, Arg, and Tyr at position 283;amino acid substitution with at position 284 is any one of Asp, Glu,Leu, Asn, Thr, and Tyr;amino acid substitution with any one of Asp, Glu, Lys, Gln, Trp, and Tyrat position 285;amino acid substitution with any one of Glu, Gly, Pro, and Tyr atposition 286;amino acid substitution with any one of Asn, Asp, Glu, and Tyr atposition 288;amino acid substitution with any one of Asp, Gly, His, Leu, Asn, Ser,Thr, Trp, and Tyr at position 290;amino acid substitution with any one of Asp, Glu, Gly, His, Ile, Gln,and Thr at position 291;amino acid substitution with any one of Ala, Asp, Glu, Pro, Thr, and Tyrat position 292;amino acid substitution with any one of Phe, Gly, His, Ile, Leu, Met,Asn, Pro, Arg, Ser, Thr, Val,Trp, and Tyr at position 293;amino acid substitution with any one of Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 294;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 295;amino acid substitution with any one of Ala, Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at position 296;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position297;amino acid substitution with any one of Ala, Asp, Glu, Phe, His, Ile,Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at position 298;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr atposition 299;amino acid substitution with any one of Ala, Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position300;amino acid substitution with any one of Asp, Glu, His, and Tyr atposition 301;amino acid substitution with Ile at position 302;amino acid substitution with any one of Asp, Gly, and Tyr at position303;amino acid substitution with at position 304 is any one of Asp, His,Leu, Asn, and Thr;amino acid substitution with any one of Glu, Ile, Thr, and Tyr atposition 305;amino acid substitution with any one of Ala, Asp, Asn, Thr, Val, and Tyrat position 311;amino acid substitution with Phe at position 313;amino acid substitution with Leu at position 315;amino acid substitution with either Glu or Gln at position 317;amino acid substitution with any one of His, Leu, Asn, Pro, Gln, Arg,Thr, Val, and Tyr at position 318;amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Leu,Asn, Pro, Ser, Thr, Val, Trp, and Tyr at position 320;amino acid substitution with any one of Ala, Asp, Phe, Gly, His, Ile,Pro, Ser, Thr, Val, Trp, and Tyr at position 322;amino acid substitution with Ile at position 323;amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Leu,Met, Pro, Arg, Thr, Val, Trp, and Tyr at position 324;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 325;amino acid substitution with any one of Ala, Asp, Glu, Gly, Ile, Leu,Met, Asn, Pro, Gln, Ser, Thr,Val, Trp, and Tyr at position 326;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at position327;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 328;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position329;amino acid substitution with any one of Cys, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position330;amino acid substitution with any one of Asp, Phe, His, Ile, Leu, Met,Gln, Arg, Thr, Val, Trp, and Tyr at position 331;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 332;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at position 333;amino acid substitution with any one of Ala, Glu, Phe, Ile, Leu, Pro,and Thr at position 334;amino acid substitution with any one of Asp, Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at position 335;amino acid substitution with any one of Glu, Lys, and Tyr at position336;amino acid substitution with any one of Glu, His, and Asn at position337;amino acid substitution with any one of Asp, Phe, Gly, Ile, Lys, Met,Asn, Gln, Arg, Ser, and Thr at position 339;amino acid substitution with either Ala or Val at position 376;amino acid substitution with either Gly or Lys at position 377;amino acid substitution with Asp at position 378;amino acid substitution with Asn at position 379;amino acid substitution with any one of Ala, Asn, and Ser at position380;amino acid substitution with either Ala or Ile at position 382;amino acid substitution with Glu at position 385;amino acid substitution with Thr at position 392;amino acid substitution with Leu at position 396;amino acid substitution with Lys at position 421;amino acid substitution with Asn at position 427;amino acid substitution with either Phe or Leu at position 428;amino acid substitution with Met at position 429;amino acid substitution with Trp at position 434;amino acid substitution with Ile at position 436; andamino acid substitution with any one of Gly, His, Ile, Leu, and Tyr atposition 440,according to EU numbering in the Fc region;[32] the method of any one of [28] to [31], wherein thenaturally-occurring Fc region is an Fc region of any one of human IgG1,human IgG2, human IgG3, and human IgG4 in which the sugar chain attachedat position 297 according to EU numbering is a fucose-containing sugarchain;[33] the method of any one of [28] to [32], comprising the step ofmodifying the Fc region so that the percentage of the Fc region to whicha fucose-deficient sugar chain is attached, or bisectingN-acetylglucosamine is added, at position 297 according to EU numberingin the Fc region, will become higher;[34] a method for producing a pharmaceutical composition which inducesan immune response, which comprises the steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain under a high calcium ion concentration condition;

(b) determining the antigen-binding activity of the antigen-bindingdomain under a low calcium ion concentration condition;

(c) selecting the antigen-binding domain whose antigen-binding activitydetermined in (a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding an FcRn-binding domainhaving FcRn-binding activity in a neutral pH range;

(e) culturing a cell into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from culture fluid of thecell cultured in (e);

[35] a method for producing a pharmaceutical composition which inducesan immune response, which comprises the steps of:

(a) determining the antigen-binding activity of an antibody under a highcalcium ion concentration condition;

(b) determining the antigen-binding activity of the antibody under a lowcalcium ion concentration condition;

(c) selecting the antibody whose antigen-binding activity determined in(a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domain of theantibody selected in (c) to a polynucleotide encoding an FcRn-bindingdomain having FcRn-binding activity in a neutral pH range;

(e) culturing a cell into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from culture fluid of thecell cultured in (e);

[36] a method for producing an antigen-binding molecule, which comprisesthe steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain in a neutral pH range;

(b) determining the antigen-binding activity of the antigen-bindingdomain in an acidic pH range;

(c) selecting the antigen-binding domain whose antigen-binding activitydetermined in (a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding an FcRn-binding domainhaving FcRn-binding activity in a neutral pH range;

(e) culturing a cell into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from culture fluid of thecell cultured in (e);

[37] a method for producing an antigen-binding molecule, which comprisesthe steps of:

(a) determining the antigen-binding activity of an antibody in a neutralpH range;

(b) determining the antigen-binding activity of the antibody in anacidic pH range;

(c) selecting the antibody whose antigen-binding activity determined in(a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domain of theantibody selected in (c) to a polynucleotide encoding an FcRn-bindingdomain having FcRn-binding activity in a neutral pH range;

(e) culturing a cell into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from culture fluid of thecell cultured in (e);

[38] the method of any one of [34] to [37], wherein the antigen-bindingmolecule has neutralizing activity against the antigen;[39] the method of any one of [34] to [38], wherein the antigen-bindingmolecule has cytotoxic activity against a cell expressing the antigen;[40] the method of any one of [34] to [39], wherein the FcRn-bindingdomain comprises an antibody Fc region;[41] the method of [40], wherein the Fc region is an Fc region in whichat least one or more amino acids selected from the group consisting ofamino acids at positions 257, 308, 428, and 434 according to EUnumbering in the Fc region are different from amino acids atcorresponding positions in a naturally-occurring Fc region;[42] the method of [40] or [41], wherein the Fc region comprises atleast one or more amino acids selected from the group consisting of:Ala at amino acid position 257;Pro at amino acid position 308;Leu at amino acid position 428; andTyr at amino acid position 434,according to EU numbering in the Fc region;[43] the method of any one of [40] to [42], wherein the Fcγreceptor-binding activity of the Fc region is higher than that of anaturally-occurring human IgG Fc region in which the sugar chainattached at position 297 according to EU numbering is afucose-containing sugar chain;[44] the method of [43], wherein the Fcγ receptor is FcγRIa, FcγRIIa(R),FcγRIIa(H), FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F);[45] the method of [43] or [44], wherein the Fc region comprises atleast one or more amino acids selected from the group consisting of:either Lys or Tyr at amino acid position 221;any one of Phe, Trp, Glu, and Tyr at amino acid position 222;any one of Phe, Trp, Glu, and Lys at amino acid position 223;any one of Phe, Trp, Glu, and Tyr at amino acid position 224;any one of Glu, Lys, and Trp at amino acid position 225;any one of Glu, Gly, Lys, and Tyr at amino acid position 227;any one of Glu, Gly, Lys, and Tyr at amino acid position 228;any one of Ala, Glu, Gly, and Tyr at amino acid position 230;any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 231;any one of Glu, Gly, Lys, and Tyr at amino acid position 232;any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 233;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 235;any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 236;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 237;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 238;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Thr, Val, Trp, and Tyr at amino acid position 239;any one of Ala, Ile, Met, and Thr at amino acid position 240;any one of Asp, Glu, Leu, Arg, Trp, and Tyr at amino acid position 241;any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at amino acid position243;His at amino acid position 244;Ala at amino acid position 245;any one of Asp, Glu, His, and Tyr at amino acid position 246;any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at aminoacid position 247;any one of Glu, His, Gln, and Tyr at amino acid position 249;either Glu or Gln at amino acid position 250;Phe at amino acid position 251;any one of Phe, Met, and Tyr at amino acid position 254;any one of Glu, Leu, and Tyr at amino acid position 255;any one of Ala, Met, and Pro at amino acid position 256;any one of Asp, Glu, His, Ser, and Tyr at amino acid position 258;any one of Asp, Glu, His, and Tyr at amino acid position 260;any one of Ala, Glu, Phe, Ile, and Thr at amino acid position 262;any one of Ala, Ile, Met, and Thr at amino acid position 263;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Trp, and Tyr at amino acid position 264;any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Val, Trp, and Tyr at amino acid position 265;any one of Ala, Ile, Met, and Thr at amino acid position 266;any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Thr, Val, Trp, and Tyr at amino acid position 267;any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr,Val, and Trp at amino acid position 268;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 269;any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr,Trp, and Tyr at amino acid position 270;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 271;any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 272;either Phe or Ile at amino acid position 273;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 274;either Leu or Trp at amino acid position 275;any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 276;any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, and Trp at amino acid position 278;Ala at amino acid position 279;any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at amino acidposition 280;any one of Asp, Lys, Pro, and Tyr at amino acid position 281;any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 282;any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at aminoacid position 283;any one of Asp, Glu, Leu, Asn, Thr, and Tyr at amino acid position 284;any one of Asp, Glu, Lys, Gln, Trp, and Tyr at amino acid position 285;any one of Glu, Gly, Pro, and Tyr at amino acid position 286;any one of Asn, Asp, Glu, and Tyr at amino acid position 288;any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at amino acidposition 290;any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at amino acid position291;any one of Ala, Asp, Glu, Pro, Thr, and Tyr at amino acid position 292;any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, and Tyr at amino acid position 293;any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 294;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 295;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, and Val at amino acid position 296;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 297;any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr,Val, Trp, and Tyr at amino acid position 298;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Val, Trp, and Tyr at amino acid position 299;any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, and Trp at amino acid position 300;any one of Asp, Glu, His, and Tyr at amino acid position 301;Ile at amino acid position 302;any one of Asp, Gly, and Tyr at amino acid position 303;any one of Asp, His, Leu, Asn, and Thr at amino acid position 304;any one of Glu, Ile, Thr, and Tyr at amino acid position 305;any one of Ala, Asp, Asn, Thr, Val, and Tyr at amino acid position 311;Phe at amino acid position 313;Leu at amino acid position 315;either Glu or Gln at amino acid position 317;any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at amino acidposition 318;any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp,and Tyr at amino acid position 320;any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, andTyr at amino acid position 322;Ile at amino acid position 323;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp,and Tyr at amino acid position 324;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 325;any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr,Val, Trp, and Tyr at amino acid position 326;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Arg, Thr, Val, Trp, and Tyr at amino acid position 327;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 329;any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 330;any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, andTyr at amino acid position 331;any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 332;any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr,Val, and Tyr at amino acid position 333;any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at amino acid position334;any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val,Trp, and Tyr at amino acid position 335;any one of Glu, Lys, and Tyr at amino acid position 336;any one of Glu, His, and Asn at amino acid position 337;any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr atamino acid position 339;either Ala or Val at amino acid position 376;either Gly or Lys at amino acid position 377;Asp at amino acid position 378;Asn at amino acid position 379;any one of Ala, Asn, and Ser at amino acid position 380;either Ala or Ile at amino acid position 382;Glu at amino acid position 385;Thr at amino acid position 392;Leu at amino acid position 396;Lys at amino acid position 421;Asn at amino acid position 427;either Phe or Leu at amino acid position 428;Met at amino acid position 429;Trp at amino acid position 434;Ile at amino acid position 436; andany one of Gly, His, Ile, Leu, and Tyr at amino acid position 440;according to EU numbering in the Fc region;[46] the method of any one of [43] to [45], wherein thenaturally-occurring Fc region is an Fc region of any one of human IgG1,human IgG2, human IgG3, and human IgG4 in which the sugar chain attachedat position 297 according to EU numbering is a fucose-containing sugarchain;[47] the method of any one of [43] to [46], wherein the Fc region ismodified so that the percentage of the Fc region to which afucose-deficient sugar chain is attached, or bisectingN-acetylglucosamine is added, at position 297 according to EU numberingin the Fc region, will become higher.

Effects of the Invention

The present invention provides pharmaceutical compositions comprising anantigen-binding molecule that, when administered to a living organism,can not only exhibit pharmacological actions on a target antigen butalso induce an immune response to the target antigen, which was notpossible with conventional antibodies, and provides methods formanufacturing them. This enables effective treatment of cancer andinfectious diseases by induction of immune response to a target antigenwhile having binding activity to the target antigen and havingcytotoxicity and antiproliferative activity against target cells, whichwas not possible with conventional vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows changes in the concentration of soluble human IL-6 receptorin mice plasma for the anti-mouse CD4 antibody administration group andthe non-administration group in a soluble human IL-6 receptorsteady-state model. The horizontal axis shows the number of days fromanti-mouse CD4 antibody administration, and the vertical axis shows theplasma concentration of soluble human IL-6 receptor.

FIG. 2 shows changes in the concentration of soluble human IL-6 receptorin mice plasma for the normal anti-IL-6 receptor antibody andpH-dependent IL-6 receptor antibody administration groups in a humanIL-6 receptor immunotolerance normal mouse model. The horizontal axisshows the number of days from anti-IL-6 receptor antibodyadministration, and the vertical axis shows the plasma concentration ofsoluble human IL-6 receptor. The filled circles indicate the plasmaconcentration of soluble human IL-6 receptor in control mice. The opencircles indicate the plasma concentration of soluble human IL-6 receptorin H54/L28-IgG1-administered mice, and the diamonds indicate the plasmaconcentration of soluble human IL-6 receptor in Fv4-IgG1-administeredmice.

FIG. 3 shows changes in the concentration of soluble human IL-6 receptorin mice for the group to which normal anti-IL-6 receptor antibody withenhanced FcRn binding at pH7.4 was administered and the group to whichpH-dependent IL-6 receptor antibody with enhanced FcRn binding at pH7.4was administered, in a human IL-6 receptor immunotolerance normal mousemodel. The horizontal axis shows the number of days from anti-IL-6receptor antibody administration, and the vertical axis shows the plasmaconcentration of soluble human IL-6 receptor. The filled circlesindicate the plasma concentration of soluble human IL-6 receptor incontrol mice. The plasma concentration of soluble human IL-6 receptor inmice to which H54/L28-IgG1, Fv4-IgG1, H54/L28-F157, or Fv4-F157 wasadministered is shown by the open circles, diamonds, open triangles, orX and the filled squares, respectively.

FIG. 4 shows a non-limiting action model of anion-concentration-dependent antigen-binding molecule with regard to thelysosome transport of a soluble antigen. Under the plasma ionconcentration conditions (in the neutral pH range or under high calciumion concentration), the antigen-binding molecule that has bound to asoluble antigen in plasma (A) is taken up into a cell by non-specificendocytosis and such (B), and then is transferred to an acidic endosomewhere it binds to FcRn expressed in the endosome via the FcRn bindingdomain under the acidic pH condition, and releases the antigen underendosomal ion concentration conditions (in the acidic pH range or underlow calcium ion concentration) (C). The released antigen is transferredto the lysosome and then degraded (D). On the other hand, theantigen-binding molecule that has released the antigen moves to the cellsurface while being bound to FcRn, dissociates from FcRn under theneutral pH condition in the plasma, and then returns to the plasma (E).

FIG. 5 shows a non-limiting action model of an antigen-binding moleculehaving FcRn-binding activity at pH7.4 with regard to the lysosometransport of a soluble antigen. Under the plasma ion concentrationconditions (in the neutral pH range or under high calcium ionconcentration), the antigen-binding molecule which has bound to asoluble antigen in plasma binds to FcRn under the neutral pH conditionvia the FcRn-binding domain (A), and this is then taken up into the cellby endocytosis (B). The antigen-binding molecule that has transferred toan acidic endosome does not release the antigen under endosomal ionconcentration conditions (in the acidic pH range or under low calciumion concentration) (C), and the antigen-bound antigen-binding moleculeis recycled onto the cell surface while being bound to FcRn (D).

FIG. 6 shows a non-limiting action model of anion-concentration-dependent antigen-binding molecule with enhanced FcRnbinding at pH7.4 with respect to lysosome transport of a solubleantigen. Under the plasma ion concentration conditions (in the neutralpH range or under high calcium ion concentration), the antigen-bindingmolecule which has bound to a soluble antigen in plasma binds to FcRnunder neutral pH conditions via the FcRn-binding domain (A), and this isthen taken up into the cell by endocytosis (B). The antigen-bindingmolecule that has transferred to an acidic endosome releases the antigenunder endosomal ion concentration conditions (in the acidic pH range orunder low calcium ion concentration) (C). The dissociated antigen istransferred to the lysosome and then degraded (D). On the other hand,the antigen-binding molecule that has released the antigen is recycledonto the cell surface while being bound to FcRn (E).

FIG. 7 shows changes in the plasma concentration of soluble human IL-6receptor and changes in the titer of mouse anti-human IL-6 receptorantibody in each of the three Fv4-F157-administered mice (#7, 8, and 9)in Test 1. The horizontal axis shows the number of days from anti-IL-6receptor antibody administration, the left vertical axis shows theconcentration of soluble human IL-6 receptor in plasma, and the rightvertical axis shows the ECL value, which serves as the indicator for themouse anti-hsIL-6R antibody titer. The solid lines show the solublehuman IL-6 receptor concentration in plasma and the dashed lines showthe ECL values. The diamonds, open squares, and triangles show changesin the plasma concentration of soluble human IL-6 receptor ofindividuals #7, 8, and 9, respectively; and X, filled squares, andfilled circles show changes in the ECL value in individuals #7, 8, and9, respectively.

FIG. 8 shows changes in the plasma concentration of soluble human IL-6receptor and changes in the titer of mouse anti-human IL-6 receptorantibody in each of the three Fv4-F157-administered mice (#10, 11, and12) of Test 2. The horizontal axis shows the number of days fromanti-IL-6 receptor antibody administration, the left vertical axis showsthe concentration of soluble human IL-6 receptor in plasma, and theright vertical axis shows the ECL value, which serves as the indicatorfor the mouse anti-human IL-6 receptor antibody titer. The solid linesshow the soluble human IL-6 receptor concentration in plasma and thedashed lines show the ECL values. The diamonds, open squares, andtriangles show the concentration of soluble human IL-6 receptor in theplasma of individuals #10, 11, and 12, respectively; and X, filledsquares, and filled circles show changes in the ECL value in individuals#10, 11, and 12, respectively.

FIG. 9 shows changes in the titer of mouse anti-hsIL-6R antibody andmouse anti-Fv4-F157 antibody in each of the three Fv4-F157-administeredmice (#7, 8, and 9) of Test 1. The horizontal axis shows the number ofdays from the administration of anti-IL-6 receptor antibody, and thevertical axis shows the ECL values, which serve as the indicators forthe mouse anti-human IL-6 receptor antibody titer and mouseanti-Fv4-F157 antibody titer. The solid lines show changes in the ECLvalue serving as the indicator for mouse anti-Fv4-F157 antibody titer,and the dashed lines show changes in the ECL value serving as theindicator for mouse anti-human IL-6 receptor antibody titer. Thediamonds, open squares, and filled triangles show changes in the ECLvalue serving as the indicator for mouse anti-Fv4-F157 antibody titer inindividuals #7, 8, and 9, respectively; and the open squares, filledsquares, and open triangles show changes in the ECL value serving as theindicator for the mouse anti-human IL-6 receptor antibody titer inindividuals #7, 8, and 9, respectively.

FIG. 10 shows changes in the titer of mouse anti-human IL-6 receptorantibody and changes in the titer of mouse anti-Fv4-F157 antibody ineach of the three Fv4-F157-administered mice (#10, 11, and 12) of Test2. The horizontal axis shows the number of days from the administrationof anti-IL-6 receptor antibody, and the vertical axis shows the ECLvalues serving as the indicator for the mouse anti-human IL-6 receptorantibody titer and the indicator for the mouse anti-Fv4-F157 antibodytiter. The solid lines show changes in the ECL value serving as theindicator for mouse anti-Fv4-F157 antibody titer, and the dashed linesshow changes in the ECL value serving as the indicator for mouseanti-hsIL-6R antibody titer. The diamonds, open squares, and filledtriangles show changes in the ECL value serving as the indicator formouse anti-Fv4-F157 antibody titer in individuals #10, 11, and 12,respectively; and the open squares, filled squares, and open trianglesshow changes in the ECL value serving as the indicator for the mouseanti-human IL-6 receptor antibody titer in individuals #10, 11, and 12,respectively.

FIG. 11 depicts a non-limiting model of the mechanism of action of anantibody fused with a target antigen on cancer cells andantigen-presenting cells.

FIG. 12 depicts a non-limiting model of the mechanism of action of anantigen-binding molecule on cancer cells and antigen-presenting cells,where the antigen-binding molecule has FcRn-binding activity in theneutral pH range and has ion concentration-dependent binding activity toa target antigen.

FIG. 13 depicts the manner of interaction between an antigen and acalcium-dependent binding antibody in plasma (2 mM CO and in endosome (3μM Ca′) (i), and the manner of interaction between an antigen and a pH-and calcium-dependent binding antibody in plasma (pH7.4, 2 mM CO and inendosome (pH6.0, 3 μM (ii).

FIG. 14 shows an ion-exchange chromatogram for an antibody comprising ahuman Vk5-2 sequence and an antibody comprising an hVk5-2_L65 sequencewhich has a modified glycosylation sequence of the human Vk5-2 sequence.The solid line represents a chromatogram for the antibody comprising thehuman Vk5-2 sequence (heavy chain: CIM_H, SEQ ID NO: 45; and lightchain: hVk5-2, SEQ ID NO: 50). The broken line represents a chromatogramfor the antibody comprising the hVk5-2_L65 sequence (heavy chain: CIM_H(SEQ ID NO: 45); and light chain: hVk5-2_L65 (SEQ ID NO: 53)).

FIG. 15 shows the relationship of a designed amino acid distribution(indicated as Design) to the amino acid distribution (indicated asLibrary) for the sequence information on 290 clones isolated from E.coli introduced with a gene library of antibodies that bind to antigensin a Ca-dependent manner. The horizontal axis indicates amino acidpositions in the Kabat numbering system. The vertical axis indicates %amino acid distribution.

FIG. 16 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RC1IgG_010, antibody 6RC1IgG_012, and antibody 6RC1IgG_019under a high calcium ion concentration (1.2 mM) condition.

FIG. 17 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RC1IgG_010, antibody 6RC1IgG_012, and antibody 6RC1IgG_019under a low calcium ion concentration (3 μM) condition.

FIG. 18 depicts the structure of heavy-chain CDR3 of an Fab fragment ofantibody 6RL#9 determined by X-ray crystallography. The heavy-chain CDR3portion in the crystal structure obtained by crystallization in thepresence of calcium ions is shown in (i), and the heavy-chain CDR3portion in the crystal structure obtained by crystallization in theabsence of calcium ions is shown in (ii).

FIG. 19 shows changes in plasma antibody concentrations in normal micefor the H54/L28-IgG1 antibody, the FH4-IgG1 antibody, and the 6RL#9-IgG1antibody.

FIG. 20 shows changes in the plasma concentration of soluble human IL-6receptor (hsIL-6R) in normal mice for the H54/L28-IgG1 antibody, theFH4-IgG1 antibody, and the 6RL#9-IgG1 antibody.

FIG. 21 shows changes in the plasma antibody concentrations in normalmice for the H54/L28-N434W antibody, the FH4-N434W antibody, and the6RL#9-N434W antibody.

FIG. 22 shows changes in the plasma concentration of soluble human IL-6receptor (hsIL-6R) in normal mice for the H54/L28-N434W antibody, theFH4-N434W antibody, and the 6RL#9-N434W antibody.

FIG. 23 shows the relationship of a designed amino acid distribution(indicated as Design) to the amino acid distribution (indicated asLibrary) for the sequence information on 132 clones isolated from E.coli introduced with a gene library of antibodies that bind to antigensin a pH-dependent manner. The horizontal axis indicates amino acidpositions in the Kabat numbering system. The vertical axis indicates %amino acid distribution.

FIG. 24 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RpH#01, antibody 6RpH#02, and antibody 6RpH#03 at pH 7.4. Thehorizontal axis shows time, and the vertical axis shows RU value.

FIG. 25 shows sensorgrams for anti-IL-6R antibody (tocilizumab),antibody 6RpH#01, antibody 6RpH#02, and antibody 6RpH#03 at pH 6.0. Thehorizontal axis shows time, and the vertical axis shows RU value.

FIG. 26 shows changes in the average plasma concentration of hsIL-6R inthe non-antibody administration group, and the Fv4-mIgG1, Fv4-mIgG2a,Fv4-mF3, Fv4-mFa30, and H54/L28-mF3 administration groups.

FIG. 27 shows changes in the antibody titer of mouse anti-human IL-6receptor antibody (anti-hsIL-6R antibody) for each individual in theFv4-mIgG1 administration group.

FIG. 28 shows changes in the antibody titer of mouse anti-human IL-6receptor antibody (anti-hsIL-6R antibody) for each individual in theFv4-mIgG2a administration group.

FIG. 29 shows changes in the antibody titer of mouse anti-human IL-6receptor antibody (anti-hsIL-6R antibody) for each individual in theFv4-mF3 administration group.

FIG. 30 shows changes in the antibody titer of mouse anti-human IL-6receptor antibody (anti-hsIL-6R antibody) for each individual in theFv4-mFa30 administration group.

FIG. 31 shows changes in the antibody titer of mouse anti-human IL-6receptor antibody (anti-hsIL-6R antibody) for each individual in theH54/L28-mF3 administration group.

FIG. 32A is a schematic diagram showing the relationship between thegenomic DNA structure of the mouse interleukin-6 receptor (Il6ra) gene(1) and the knock-in vector to be inserted (2). The knock-in vector hasthe full-length human interleukin-6-receptor (hIL6R) cDNA, the hp7sequence, a poly-A addition signal, and a neomycin-resistance gene.

FIG. 32B is a schematic diagram showing how the genomic DNA of the mouseinterleukin-6 receptor gene (a) and the knock-in vector (b) undergohomologous recombination to form a knock-in genomic DNA (c).Furthermore, it shows the process of completing the human interleukin-6receptor gene knock-in allele (d) by allowing Cre recombinase to act on(c) to remove the neomycin-resistance gene cassette. The arrows in thefigure indicate the positions for setting primers used for detecting theknocked-in human interleukin-6 receptor gene.

FIG. 33 shows a representative example of PCR which analyzed eachgenotype obtained in the process of establishing the human interleukin-6receptor gene knock-in mice.

FIG. 34 shows the expression profile of the interleukin-6 receptor genein the wild-type mouse and the human interleukin-6 receptor geneknock-in mouse.

FIG. 35 is a graph showing the results of measuring the plasmaconcentration of soluble human interleukin-6 receptor (hsIL-6R) inwild-type mice and homozygous and heterozygous human interleukin-6receptor gene knock-in mice. KI/KI, KI/+, and +/+ indicate thehomozygous knock-in mice, heterozygous knock-in mice, and the wild-type,respectively.

FIG. 36 is a graph showing the species-specific reactivity tointerleukin-6 (ligand) in the wild-type mice and homozygous humaninterleukin-6 receptor gene knock-in mice.

MODE FOR CARRYING OUT THE INVENTION

The definitions and detailed description below are provided to help theunderstanding of the present invention illustrated herein.

Amino Acids

Herein, amino acids are described in one- or three-letter codes or both,for example, Ala/A, Leu/L, Arg/R, Lys/K, Asn/N, Met/M, Asp/D, Phe/F,Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T, Gly/G Trp/W, His/H, Tyr/Y,Ile/I, or Val/V.

Antigen

Herein, an antigen is not particularly limited, and may be any antigenas long as it is a molecule that may induce the immune system of anorganism and thereby become a target of the immune response in thatorganism. Preferred examples of such antigens include molecules that areexpressed specifically in tumor cells but not expressed in normal cells(neoepitopes). Molecules that are expressed in foreign biologicalspecies such as bacteria and viruses that infect an organism but notexpressed in the organism are also preferred. The phrase “expressedspecifically in tumor cells but not expressed in normal cells” or“expressed in foreign biological species that infect an organism but notexpressed in the organism” means that there is a qualitative orquantitative difference in the molecule between “tumor cells and normalcells” or “foreign biological species that infect an organism and theorganism”. For example, even if a molecule is expressed in normal cells,if the amount of the molecule expressed in tumor cells is far greaterthan the amount expressed in the normal cells, it can be said in thepresent invention that there is a quantitative difference in themolecule between tumor cells and normal cells. Furthermore, even if theexpression level of a polypeptide consisting of the same amino acidsequence is similar in tumor cells and normal cells, if the expressedpolypeptide has undergone posttranslational modification such asphosphorylation in tumor cells but not in normal cells, it can be saidin the present invention that there is a qualitative difference in themolecule between tumor cells and normal cells.

For such molecules, preferred tumor antigens may include: ALK receptor(pleiotrophin receptor), pleiotrophin; KS 1/4 pancreas carcinomaantigen; ovarian carcinoma antigen (CA125); prostatic acid phosphate;prostate specific antigen (PSA); melanoma-associated antigen p97;melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA);prostate specific membrane antigen; carcinoembryonic antigen (CEA);polymorphic epithelial mucin antigen; human milk fat globule antigen;colorectal tumor-associated antigens such as CEA, TAG-72, CO17-1A, GICA19-9, CTA-1, and LEA; Burkitt's lymphoma antigen-38.13; CD19; humanB-lymphoma antigen-CD20; CD33; melanoma specific antigens such asganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3;tumor-specific transplantation-type cell-surface antigen (TSTA);virally-induced tumor antigens including T-antigen, DNA tumor virusesand Envelope antigens of RNA tumor viruses; oncofetal antigens such asCEA of colon, 5T4 oncofetal trophoblast glycoprotein, and bladder tumoroncofetal antigen; alpha-fetoprotein; differentiation antigens such ashuman lung carcinoma antigens L6 and L20; antigens of fibrosarcoma;human leukemia T cell antigen-Gp37; neoglycoprotein; sphingolipids;breast cancer antigens such as EGFR (epidermal growth factor receptor);NY-BR-16; NY-BR-16 and HER2 antigen (p185HER2); polymorphic epithelialmucin (PEM) antigen; malignant human lymphocyte antigen-APO-1;differentiation antigens such as I antigen found in fetal erythrocytes;primary endoderm I antigen found in adult erythrocytes; preimplantationembryos; I(Ma) found in gastric cancer; M18, M39 found in mammaryepithelium; SSEA-1 found in myeloid cells; VEP8; VEP9; Myl; VIM-D5;D156-22 found in colorectal cancer; TRA-1-85 (blood group H); SCP-1found in testis and ovarian cancer; C14 found in colon cancer; F3 foundin lung cancer; AH6 found in gastric cancer; Y hapten; Ley found inembryonal carcinoma cells; TL5 (blood group A); EGF receptor found inA431 cells; E1 series (blood group B) found in pancreatic cancer; FC10.2found in embryonal carcinoma cells; gastric cancer antigen; CO-514(blood group Lea) found in adenocarcinomas; NS-10 found inadenocarcinomas; CO-43 (blood group Leb); G49 found in EGF receptor ofA431 cells; MH2 (blood group ALeb/Ley) found in colon cancer; 19.9 foundin colon cancer; gastric cancer mucins; T5A7 found in myeloid cells; R24found in melanoma; 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, andM1:22:25:8 found in embryonal carcinoma cells and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos; subcutaneous T cell lymphomaantigen; MART-1 antigen; sialyl Tn (STn) antigen; colon cancer antigenNY-CO-45; lung cancer antigen NY-LU-12 variant A; adenocarcinoma antigenART1; paraneoplastic associated brain-testis-cancer antigen(onconeuronal antigen MA2; paraneoplastic neuronal antigen);Neuro-oncological ventral antigen 2 (NOVA2); hemocyte carcinoma antigengene 520; tumor-associated antigen CO-029; tumor-associated antigensMAGE-C1 (cancer/testis antigen CT7), MAGE-B 1 (MAGE-XP antigen), MAGE-B2(DAM6), MAGE-2, MAGE-4a, MAGE-4b and MAGE-X2; Cancer-Testis Antigen(NY-EOS-1); YKL-40, and fragments of any of the aforementionedpolypeptides or structures produced by modifying them (for example,modified phosphate group or sugar chain of the above-mentioned).

Antigens of foreign biological species include molecules expressed in:Bacillus anthraces, Clostridium botulinum, Yersinia pestis, Variolamajor (smallpox) and other poxviruses, Francisella tularensis(tularemia), and those that cause viral hemorrhagic fever, Arenavirusessuch as LCM, Junin virus, Machupo virus, Guanarito virus, and those thatcause Lassa fever, Bunyaviruses and Hantaviruses such as those thatcause Rift-valley fever, Calicivirus, hepatitis A, hepatitis B,hepatitis C, viral encephalitis such as West Nile Virus, LaCrosse,California encephalitis, VEE, EEE, WEE, and Japanese encephalitis virus,Kyasanur forest virus, tickborne hemorrhagic fever virus, Crimean-Congohemorrhagic fever virus, tickborne encephalitis viruses, Yellow fever,multidrug-resistant TB, influenza, other rickettsiae and rabies,Flavirus, Dengue, Filovirus, Ebola, Marburg Burkholderia pseudomallei,Coxiella burnetii (Q fever), Brucella species (brucellosis),Burkholderia mallei (glanders), ricin toxin (derived from Ricinuscommunis), epsilon toxin of Clostridium perfringens, Staphylococcusenterotoxin B, Typhus fever (Rickettsia prowazekii), food andwater-borne pathogens, bacteria such as diarrheagenic E. coli,pathogenic Vibrios, Shigella species, Salmonella, Listeriamonocytogenes, Campylobacter jejuni, and Yersinia enterocolitica; andprotozoas such as Cryptosporidium parvum, Cyclospora cayatanensis,Giardia lamblia, Entamoeba histolytica, Toxoplasma, and Microsporidia.

Other antigens include, for example, the molecules below: 17-IA, 4-1BB,4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33,ACE, ACE-2, activin, activin A, activin AB, activin B, activin C,activin RIA, activin RIAALK-2, activin RIB ALK-4, activin RIIA, activinRIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS,ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrialnatriuretic peptide, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R,Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik,BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP,b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA,BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer associatedantigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D,cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S,cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC,CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4,CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7,CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16,CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumorassociated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatoryfactor (Decay accelerating factor), des (1-3)-IGF-I (brain IGF-1), Dhh,digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2,EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor,enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2/EphB4, EPO, ERCC,E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX,fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF,FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3,Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3,FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2,GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13,CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15(MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3,GITR, glucagon, Glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF,gp130, gp72, GRO, growth hormone releasing hormone, hapten (NP-cap orNIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelopeglycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120,heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4),herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA,high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120,HIV MB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG Hrk, humancardiac myosin, human cytomegalovirus (HCMV), human growth hormone(HGH), HVEM, 1-309, TAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgAreceptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II,IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R,IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon(INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain,insulin B chain, insulin-like growth factor1, integrin alpha2, integrinalpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha4/beta7,integrin alpha5 (alpha V), integrin alpha5/beta1, integrin alpha5/beta3,integrin alpha6, integrin beta1, integrin beta2, interferon gamma,IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1,kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocytegrowth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1,latent TGF-1 bp1, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Yassociated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX,LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface,luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG,MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES,MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIP, MIP-1-alpha, MK, MMAC1,MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2,MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1),MUC18, Mullerian-inhibiting substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6,neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS,Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95,PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin,PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN,PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14,proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specificmembrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL,RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratorysyncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2,RSK, 5100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR,SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II,TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3,T-cell receptor (for example, T-cell receptor alpha/beta), TdT, TECK,TEM1, TEM5, TEM7, TEM8, TERT, testis PLAP-like alkaline phosphatase,TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-betaRI(ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2,TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1,thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2,Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI,TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5,KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand,TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferrin receptor, TRF, Trk, TROP-2, TSG; TSLP, tumor associatedantigen CA125, tumor associated antigen expressing Lewis-Y associatedcarbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17/IL-17R,IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1,Chromogranin A, Chromogranin B, tau, VAP1, high molecular weightkininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5,Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b,C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B,factor D, factor H, properdin, sclerostin, fibrinogen, fibrin,prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII,factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X,factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII,factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA,plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2,Syndecan-3, Syndecan-4, LPA, SIP, Acetylcholine receptor, AdipoR1,AdipoR2, ADP ribosyl cyclase-1, alpha-4/beta-7 integrin, alpha-5/beta-1integrin, alpha-v/beta-6 integrin, alphavbeta1 integrin, Angiopoietinligand-2, Angptl2, Anthrax, Cadherin, Carbonic anhydrase-IX, CD105,CD155, CD158a, CD37, CD49b, CD51, CD70, CD72, Claudin 18, Clostridiumdifficile toxin, CS1, Delta-like protein ligand 4, DHICA oxidase,Dickkopf-1 ligand, Dipeptidyl peptidase IV, EPOR, F protein of RSV,Factor Ia, FasL, Folate receptor alpha, Glucagon receptor, Glucagon-likepeptide 1 receptor, Glutamate carboxypeptidase II, GMCSFR, Hepatitis Cvirus E2 glycoprotein, Hepcidin, IL-17 receptor, IL-22 receptor, IL-23receptor, IL-3 receptor, Kit tyrosine kinase, Leucine RichAlpha-2-Glycoprotein 1 (LRG1), Lysosphingolipid receptor, Membraneglycoprotein OX2, Mesothelin, MET, MICA, MUC-16, Myelin associatedglycoprotein, Neuropilin-1, Neuropilin-2, Nogo receptor, PLXNA1, PLXNA2,PLXNA3, PLXNA4A, PLXNA4B, PLXNB1, PLXNB2, PLXNB3, PLXNC1, PLXND1,Programmed cell death ligand 1, Proprotein convertase PC9, P-selectinglycoprotein ligand-1, RAGE, Reticulon 4, RF, RON-8, SEMA3A, SEMA3B,SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SEMA4A, SEMA4B, SEMA4C, SEMA4D,SEMA4F, SEMA4G, SEMA5A, SEMA5B, SEMA6A, SEMA6B, SEMA6C, SEMA6D, SEMA7A,Shiga like toxin II, Sphingosine-1-phosphate receptor-1, ST2,Staphylococcal lipoteichoic acid, Tenascin, TG2, Thymic stromallymphoprotein receptor, TNF superfamily receptor 12A, Transmembraneglycoprotein NMB, TREM-1, TREM-2, Trophoblast glycoprotein, TSHreceptor, TTR, Tubulin, and ULBP2; and receptors for hormone and growthfactors.

Epitope

“Epitope” means an antigenic determinant in an antigen, and refers to anantigen site to which the antigen-binding domain of an antigen-bindingmolecule disclosed herein binds. Thus, for example, the epitope can bedefined according to its structure. Alternatively, the epitope may bedefined according to the antigen-binding activity of an antigen-bindingmolecule that recognizes the epitope. When the antigen is a peptide orpolypeptide, the epitope can be specified by the amino acid residuesforming the epitope. Alternatively, when the epitope is a sugar chain,the epitope can be specified by its specific sugar chain structure.

A linear epitope is an epitope that contains an epitope whose primaryamino acid sequence is recognized. Such a linear epitope typicallycontains at least three and most commonly at least five, for example,about 8 to 10 or 6 to 20 amino acids in its specific sequence.

In contrast to the linear epitope, “conformational epitope” is anepitope in which the primary amino acid sequence containing the epitopeis not the only determinant of the recognized epitope (for example, theprimary amino acid sequence of a conformational epitope is notnecessarily recognized by an epitope-defining antibody). Conformationalepitopes may contain a greater number of amino acids compared to linearepitopes. A conformational epitope-recognizing antibody recognizes thethree-dimensional structure of a peptide or protein.

For example, when a protein molecule folds and forms a three-dimensionalstructure, amino acids and/or polypeptide main chains that form aconformational epitope become aligned, and the epitope is maderecognizable by the antibody. Methods for determining epitopeconformations include, for example, X ray crystallography,two-dimensional nuclear magnetic resonance, site-specific spin labeling,and electron paramagnetic resonance, but are not limited thereto. See,for example, Epitope Mapping Protocols in Methods in Molecular Biology(1996), Vol. 66, Morris (ed.).

Binding Activity

Examples of a method for assessing the epitope binding by a testantigen-binding molecule containing an IL-6 receptor antigen-bindingdomain are described below. According to the examples below, methods forassessing the epitope binding by a test antigen-binding moleculecontaining an antigen-binding domain for an antigen other than IL-6receptor, can also be appropriately conducted.

For example, whether a test antigen-binding molecule containing an IL-6receptor antigen-binding domain recognizes a linear epitope in the IL-6receptor molecule can be confirmed for example as mentioned below. Alinear peptide comprising an amino acid sequence forming theextracellular domain of IL-6 receptor is synthesized for the abovepurpose. The peptide can be synthesized chemically, or obtained bygenetic engineering techniques using a region encoding the amino acidsequence corresponding to the extracellular domain in an IL-6 receptorcDNA. Then, a test antigen-binding molecule containing an IL-6 receptorantigen-binding domain is assessed for its binding activity towards alinear peptide comprising the amino acid sequence forming theextracellular domain. For example, an immobilized linear peptide can beused as an antigen by ELISA to evaluate the binding activity of theantigen-binding molecule towards the peptide. Alternatively, the bindingactivity towards a linear peptide can be assessed based on the levelthat the linear peptide inhibits the binding of the antigen-bindingmolecule to IL-6 receptor-expressing cells. These tests can demonstratethe binding activity of the antigen-binding molecule towards the linearpeptide.

Whether a test antigen-binding molecule containing an IL-6 receptorantigen-binding domain recognizes a conformational epitope can beassessed as follows. IL-6 receptor-expressing cells are prepared for theabove purpose. A test antigen-binding molecule containing an IL-6receptor antigen-binding domain can be determined to recognize aconformational epitope when it strongly binds to IL-6receptor-expressing cells upon contact, but does not substantially bindto an immobilized linear peptide comprising an amino acid sequenceforming the extracellular domain of IL-6 receptor. Herein, “notsubstantially bind” means that the binding activity is 80% or less,generally 50% or less, preferably 30% or less, and particularlypreferably 15% or less compared to the binding activity towards cellsexpressing human IL-6 receptor.

Methods for assaying the binding activity of a test antigen-bindingmolecule containing an IL-6 receptor antigen-binding domain towards IL-6receptor-expressing cells include, for example, the methods described inAntibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold SpringHarbor Laboratory (1988) 359-420). Specifically, the assessment can beperformed based on the principle of ELISA or fluorescence activated cellsorting (FACS) using IL-6 receptor-expressing cells as antigen.

In the ELISA format, the binding activity of a test antigen-bindingmolecule containing an IL-6 receptor antigen-binding domain towards IL-6receptor-expressing cells can be assessed quantitatively by comparingthe levels of signal generated by enzymatic reaction. Specifically, atest polypeptide complex is added to an ELISA plate onto which IL-6receptor-expressing cells are immobilized. Then, the testantigen-binding molecule bound to the cells is detected using anenzyme-labeled antibody that recognizes the test antigen-bindingmolecule. Alternatively, when FACS is used, a dilution series of a testantigen-binding molecule is prepared, and the antibody binding titer forIL-6 receptor-expressing cells can be determined to compare the bindingactivity of the test antigen-binding molecule towards IL-6receptor-expressing cells.

The binding of a test antigen-binding molecule towards an antigenexpressed on the surface of cells suspended in buffer or the like can bedetected using a flow cytometer. Known flow cytometers include, forexample, the following devices:

FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE

FACSCalibur™ (all are trade names of BD Biosciences)

EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC

Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of BeckmanCoulter).

Preferable methods for assaying the binding activity of a testantigen-binding molecule containing an IL-6 receptor antigen-bindingdomain towards an antigen include, for example, the following method.First, IL-6 receptor-expressing cells are reacted with a testantigen-binding molecule, and then this is stained with an FITC-labeledsecondary antibody that recognizes the antigen-binding molecule. Thetest antigen-binding molecule is appropriately diluted with a suitablebuffer to prepare the molecule at a desired concentration. For example,the molecule can be used at a concentration within the range of 10 μg/mlto 10 ng/ml. Then, the fluorescence intensity and cell count aredetermined using FACSCalibur (BD). The fluorescence intensity obtainedby analysis using the CELL QUEST Software (BD), i.e., the Geometric Meanvalue, reflects the quantity of antibody bound to cells. That is, thebinding activity of a test antigen-binding molecule, which isrepresented by the quantity of the test antigen-binding molecule bound,can be determined by measuring the Geometric Mean value.

Whether a test antigen-binding molecule containing an IL-6 receptorantigen-binding domain shares a common epitope with anotherantigen-binding molecule can be assessed based on the competitionbetween the two molecules for the same epitope. The competition betweenantigen-binding molecules can be detected by cross-blocking assay or thelike. For example, the competitive ELISA assay is a preferredcross-blocking assay.

Specifically, in cross-blocking assay, the IL-6 receptor proteinimmobilized to the wells of a microtiter plate is pre-incubated in thepresence or absence of a candidate competitor antigen-binding molecule,and then a test antigen-binding molecule is added thereto. The quantityof test antigen-binding molecule bound to the IL-6 receptor protein inthe wells is indirectly correlated with the binding ability of acandidate competitor antigen-binding molecule that competes for thebinding to the same epitope. That is, the greater the affinity of thecompetitor antigen-binding molecule for the same epitope, the lower thebinding activity of the test antigen-binding molecule towards the IL-6receptor protein-coated wells.

The quantity of the test antigen-binding molecule bound to the wells viathe IL-6 receptor protein can be readily determined by labeling theantigen-binding molecule in advance. For example, a biotin-labeledantigen-binding molecule is measured using an avidin/peroxidaseconjugate and appropriate substrate. In particular, cross-blocking assaythat uses enzyme labels such as peroxidase is called “competitive ELISAassay”. The antigen-binding molecule can also be labeled with otherlabeling substances that enable detection or measurement. Specifically,radiolabels, fluorescent labels, and such are known.

When the candidate competitor antigen-binding molecule can block thebinding by a test antigen-binding molecule containing an IL-6 receptorantigen-binding domain by at least 20%, preferably at least 20 to 50%,and more preferably at least 50% compared to the binding activity in acontrol experiment conducted in the absence of the competitorantigen-binding molecule, the test antigen-binding molecule isdetermined to substantially bind to the same epitope bound by thecompetitor antigen-binding molecule, or compete for the binding to thesame epitope.

When the structure of an epitope bound by a test antigen-bindingmolecule containing an IL-6 receptor antigen-binding domain has alreadybeen identified, whether the test and control antigen-binding moleculesshare a common epitope can be assessed by comparing the bindingactivities of the two antigen-binding molecules towards a peptideprepared by introducing amino acid mutations into the peptide formingthe epitope.

To measure the above binding activities, for example, the bindingactivities of test and control antigen-binding molecules towards alinear peptide into which a mutation is introduced are compared in theabove ELISA format. Besides the ELISA methods, the binding activitytowards the mutant peptide bound to a column can be determined byflowing test and control antigen-binding molecules in the column, andthen quantifying the antigen-binding molecule eluted in the elutionsolution. Methods for adsorbing a mutant peptide to a column, forexample, in the form of a GST fusion peptide, are known.

Alternatively, when the identified epitope is a conformational epitope,whether test and control antigen-binding molecules share a commonepitope can be assessed by the following method. First, IL-6receptor-expressing cells and cells expressing IL-6 receptor with amutation introduced into the epitope are prepared. The test and controlantigen-binding molecules are added to a cell suspension prepared bysuspending these cells in an appropriate buffer such as PBS. Then, thecell suspensions are appropriately washed with a buffer, and anFITC-labeled antibody that recognizes the test and controlantigen-binding molecules is added thereto. The fluorescence intensityand number of cells stained with the labeled antibody are determinedusing FACSCalibur (BD). The test and control antigen-binding moleculesare appropriately diluted using a suitable buffer, and used at desiredconcentrations. For example, they may be used at a concentration withinthe range of 10 μg/ml to 10 ng/ml. The fluorescence intensity determinedby analysis using the CELL QUEST Software (BD), i.e., the Geometric Meanvalue, reflects the quantity of labeled antibody bound to cells. Thatis, the binding activities of the test and control antigen-bindingmolecules, which are represented by the quantity of labeled antibodybound, can be determined by measuring the Geometric Mean value.

In the above method, whether an antigen-binding molecule does “notsubstantially bind to cells expressing mutant IL-6 receptor” can beassessed, for example, by the following method. First, the test andcontrol antigen-binding molecules bound to cells expressing mutant IL-6receptor are stained with a labeled antibody. Then, the fluorescenceintensity of the cells is determined. When FACSCalibur is used forfluorescence detection by flow cytometry, the determined fluorescenceintensity can be analyzed using the CELL QUEST Software. From theGeometric Mean values in the presence and absence of the polypeptidecomplex, the comparison value (ΔGeo-Mean) can be calculated according tothe following formula to determine the ratio of increase in fluorescenceintensity as a result of the binding by the antigen-binding molecule.

ΔGeo-Mean=Geo-Mean (in the presence of the polypeptide complex)/Geo-Mean(in the absence of the polypeptide complex)

The Geometric Mean comparison value (ΔGeo-Mean value for the mutant IL-6receptor molecule) determined by the above analysis, which reflects thequantity of a test antigen-binding molecule bound to cells expressingmutant IL-6 receptor, is compared to the ΔGeo-Mean comparison value thatreflects the quantity of the test antigen-binding molecule bound to IL-6receptor-expressing cells. In this case, the concentrations of the testantigen-binding molecule used to determine the ΔGeo-Mean comparisonvalues for IL-6 receptor-expressing cells and cells expressing mutantIL-6 receptor are particularly preferably adjusted to be equal orsubstantially equal. An antigen-binding molecule that has been confirmedto recognize an epitope in IL-6 receptor is used as a controlantigen-binding molecule.

If the ΔGeo-Mean comparison value of a test antigen-binding molecule forcells expressing mutant IL-6 receptor is smaller than the ΔGeo-Meancomparison value of the test antigen-binding molecule for IL-6receptor-expressing cells by at least 80%, preferably 50%, morepreferably 30%, and particularly preferably 15%, then the testantigen-binding molecule “does not substantially bind to cellsexpressing mutant IL-6 receptor”. The formula for determining theGeo-Mean (Geometric Mean) value is described in the CELL QUEST SoftwareUser's Guide (BD biosciences). When the comparison shows that thecomparison values are substantially equivalent, the epitope for the testand control antigen-binding molecules can be determined to be the same.

Antigen-Binding Domain

An “antigen-binding domain” may be of any structure as long as it bindsto an antigen of interest. Such domains preferably include, for example:

antibody heavy-chain and light-chain variable regions;a module of about 35 amino acids called A domain which is contained inthe in vivo cell membrane protein Avimer (WO 2004/044011, WO2005/040229);Adnectin containing the 10Fn3 domain which binds to the protein moietyof fibronectin, a glycoprotein expressed on cell membrane (WO2002/032925);Affibody which is composed of a 58-amino acid three-helix bundle basedon the scaffold of the IgG-binding domain of Protein A (WO 1995/001937);Designed Ankyrin Repeat proteins (DARPins) which are a region exposed onthe molecular surface of ankyrin repeats (AR) having a structure inwhich a subunit consisting of a turn comprising 33 amino acid residues,two antiparallel helices, and a loop is repeatedly stacked (WO2002/020565);Anticalins and such, which are domains consisting of four loops thatsupport one side of a barrel structure composed of eight circularlyarranged antiparallel strands that are highly conserved among lipocalinmolecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003/029462); andthe concave region formed by the parallel-sheet structure inside thehorseshoe-shaped structure constituted by stacked repeats of theleucine-rich-repeat (LRR) module of the variable lymphocyte receptor(VLR) which does not have the immunoglobulin structure and is used inthe system of acquired immunity in jawless vertebrate such as lamperyand hagfish (WO 2008/016854). Preferred antigen-binding domains of thepresent invention include, for example, those having antibodyheavy-chain and light-chain variable regions. Preferred examples ofantigen-binding domains include “single chain Fv (scFv)”, “single chainantibody”, “Fv”, “single chain Fv 2 (scFv2)”, “Fab”, and “F(ab′)2”.

The antigen-binding domains of antigen-binding molecules of the presentinvention can bind to an identical epitope. Such epitope can be present,for example, in a protein comprising the amino acid sequence of SEQ IDNO: 1. Alternatively, each of the antigen-binding domains ofantigen-binding molecules of the present invention can bind to adifferent epitope. Herein, the different epitope can be present in, forexample, a protein comprising the amino acid sequence of SEQ ID NO: 1.

Specificity

“Specific” means that one of molecules that specifically binds to doesnot show any significant binding to molecules other than a single or anumber of binding partner molecules. Furthermore, “specific” is alsoused when an antigen-binding domain is specific to a particular epitopeamong multiple epitopes in an antigen. When an epitope bound by anantigen-binding domain is contained in multiple different antigens,antigen-binding molecules containing the antigen-binding domain can bindto various antigens that have the epitope.

Antibodies

Herein, “antibody” refers to a natural immunoglobulin or animmunoglobulin produced by partial or complete synthesis. Antibodies canbe isolated from natural sources such as naturally-occurring plasma andserum, or culture supernatants of antibody-producing hybridomas.Alternatively, antibodies can be partially or completely synthesizedusing techniques such as genetic recombination. Preferred antibodiesinclude, for example, antibodies of an immunoglobulin isotype orsubclass belonging thereto. Known human immunoglobulins includeantibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies ofthe present invention include IgG1, IgG2, IgG3, and IgG4.

Methods for producing an antibody with desired binding activity areknown to those skilled in the art. Below is an example that describes amethod for producing an antibody that binds to IL-6 receptor (anti-IL-6receptor antibody). Antibodies that bind to an antigen other than IL-6receptor can also be produced according to the example described below.

Anti-IL-6 receptor antibodies can be obtained as polyclonal ormonoclonal antibodies using known methods. The anti-IL-6 receptorantibodies preferably produced are monoclonal antibodies derived frommammals. Such mammal-derived monoclonal antibodies include antibodiesproduced by hybridomas or host cells transformed with an expressionvector carrying an antibody gene by genetic engineering techniques.“Humanized antibodies” or “chimeric antibodies” are included in themonoclonal antibodies of the present invention.

Monoclonal antibody-producing hybridomas can be produced using knowntechniques, for example, as described below. Specifically, mammals areimmunized by conventional immunization methods using an IL-6 receptorprotein as a sensitizing antigen. Resulting immune cells are fused withknown parental cells by conventional cell fusion methods. Then,hybridomas producing an anti-IL-6 receptor antibody can be selected byscreening for monoclonal antibody-producing cells using conventionalscreening methods.

Specifically, monoclonal antibodies are prepared as mentioned below.First, the IL-6 receptor gene whose nucleotide sequence is disclosed inSEQ ID NO: 2 can be expressed to produce an IL-6 receptor protein shownin SEQ ID NO: 1, which will be used as a sensitizing antigen forantibody preparation. That is, a gene sequence encoding IL-6 receptor isinserted into a known expression vector, and appropriate host cells aretransformed with this vector. The desired human IL-6 receptor protein ispurified from the host cells or their culture supernatants by knownmethods. In order to obtain soluble IL-6 receptor from culturesupernatants, for example, a protein consisting of the amino acids atpositions 1 to 357 in the IL-6 receptor polypeptide sequence of SEQ IDNO: 1, such as described in Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968), is expressed as a soluble IL-6 receptor, instead ofthe IL-6 receptor protein of SEQ ID NO: 1. Purified natural IL-6receptor protein can also be used as a sensitizing antigen.

The purified IL-6 receptor protein can be used as a sensitizing antigenfor immunization of mammals. A partial IL-6 receptor peptide may also beused as a sensitizing antigen. In this case, a partial peptide can beprepared by chemical synthesis based on the amino acid sequence of humanIL-6 receptor, or by inserting a partial IL-6 receptor gene into anexpression vector for expression. Alternatively, a partial peptide canbe produced by degrading an IL-6 receptor protein with a protease. Thelength and region of the partial IL-6 receptor peptide are not limitedto particular embodiments. A preferred region can be arbitrarilyselected from the amino acid sequence at amino acid positions 20 to 357in the amino acid sequence of SEQ ID NO: 1. The number of amino acidsforming a peptide to be used as a sensitizing antigen is preferably atleast five or more, six or more, or seven or more. More specifically, apeptide of 8 to 50 residues, more preferably 10 to 30 residues can beused as a sensitizing antigen.

For sensitizing antigen, alternatively it is possible to use a fusionprotein prepared by fusing a desired partial polypeptide or peptide ofthe IL-6 receptor protein with a different polypeptide. For example,antibody Fc fragments and peptide tags are preferably used to producefusion proteins to be used as sensitizing antigens. Vectors forexpression of such fusion proteins can be constructed by fusing in framegenes encoding two or more desired polypeptide fragments and insertingthe fusion gene into an expression vector as described above. Methodsfor producing fusion proteins are described in Molecular Cloning 2nd ed.(Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) ColdSpring Harbor Lab. Press). Methods for preparing IL-6 receptor to beused as a sensitizing antigen, and immunization methods using IL-6receptor are specifically described in WO 2003/000883, WO 2004/022754,WO 2006/006693, and such.

There is no particular limitation on the mammals to be immunized withthe sensitizing antigen. However, it is preferable to select the mammalsby considering their compatibility with the parent cells to be used forcell fusion. In general, rodents such as mice, rats, and hamsters,rabbits, and monkeys are preferably used.

The above animals are immunized with a sensitizing antigen by knownmethods. Generally performed immunization methods include, for example,intraperitoneal or subcutaneous injection of a sensitizing antigen intomammals. Specifically, a sensitizing antigen is appropriately dilutedwith PBS (Phosphate-Buffered Saline), physiological saline, or the like.If desired, a conventional adjuvant such as Freund's complete adjuvantis mixed with the antigen, and the mixture is emulsified. Then, thesensitizing antigen is administered to a mammal several times at 4- to21-day intervals. Appropriate carriers may be used in immunization withthe sensitizing antigen. In particular, when a low-molecular-weightpartial peptide is used as the sensitizing antigen, it is sometimesdesirable to couple the sensitizing antigen peptide to a carrier proteinsuch as albumin or keyhole limpet hemocyanin for immunization.

Alternatively, hybridomas producing a desired antibody can be preparedusing DNA immunization as mentioned below. DNA immunization is animmunization method that confers immunostimulation by expressing asensitizing antigen in an animal immunized as a result of administeringa vector DNA constructed to allow expression of an antigenprotein-encoding gene in the animal. As compared to conventionalimmunization methods in which a protein antigen is administered toanimals to be immunized, DNA immunization is expected to be superior inthat:

immunostimulation can be provided while retaining the structure of amembrane protein such as IL-6 receptor; and

there is no need to purify the antigen for immunization.

In order to prepare a monoclonal antibody of the present invention usingDNA immunization, first, a DNA expressing an IL-6 receptor protein isadministered to an animal to be immunized. The IL-6 receptor-encodingDNA can be synthesized by known methods such as PCR. The obtained DNA isinserted into an appropriate expression vector, and then this isadministered to an animal to be immunized. Preferably used expressionvectors include, for example, commercially-available expression vectorssuch as pcDNA3.1. Vectors can be administered to an organism usingconventional methods. For example, DNA immunization is performed byusing a gene gun to introduce expression vector-coated gold particlesinto cells in the body of an animal to be immunized. Antibodies thatrecognized IL-6 receptor can also be produced by the methods describedin WO 2003/104453.

After immunizing a mammal as described above, an increase in the titerof an IL-6 receptor-binding antibody is confirmed in the serum. Then,immune cells are collected from the mammal, and then subjected to cellfusion. In particular, splenocytes are preferably used as immune cells.

A mammalian myeloma cell is used as a cell to be fused with theabove-mentioned immune cells. The myeloma cells preferably comprise asuitable selection marker for screening. A selection marker conferscharacteristics to cells for their survival (or death) under a specificculture condition. Hypoxanthine-guanine phosphoribosyltransferasedeficiency (hereinafter abbreviated as HGPRT deficiency) and thymidinekinase deficiency (hereinafter abbreviated as TK deficiency) are knownas selection markers. Cells with HGPRT or TK deficiency havehypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviatedas HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in a HATselection medium, and are thus killed. However, when the cells are fusedwith normal cells, they can continue DNA synthesis using the salvagepathway of the normal cells, and therefore they can grow even in the HATselection medium.

HGPRT-deficient and TK-deficient cells can be selected in a mediumcontaining 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG),or 5′-bromodeoxyuridine, respectively. Normal cells are killed becausethey incorporate these pyrimidine analogs into their DNA. Meanwhile,cells that are deficient in these enzymes can survive in the selectionmedium, since they cannot incorporate these pyrimidine analogs. Inaddition, a selection marker referred to as G418 resistance provided bythe neomycin-resistant gene confers resistance to 2-deoxystreptamineantibiotics (gentamycin analogs). Various types of myeloma cells thatare suitable for cell fusion are known.

For example, myeloma cells including the following cells can bepreferably used:

P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550); P3x63Ag8U.1(Current Topics in Microbiology and Immunology (1978) 81, 1-7);

NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519);

MPC-11 (Cell (1976) 8 (3), 405-415); SP2/0 (Nature (1978) 276 (5685),269-270);

FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);S194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);

R210 (Nature (1979) 277 (5692), 131-133), etc.

Cell fusions between the immunocytes and myeloma cells are essentiallycarried out using known methods, for example, a method by Kohler andMilstein et al. (Methods Enzymol. (1981) 73: 3-46).

More specifically, cell fusion can be carried out, for example, in aconventional culture medium in the presence of a cell fusion-promotingagent. The fusion-promoting agents include, for example, polyethyleneglycol (PEG) and Sendai virus (HVJ). If required, an auxiliary substancesuch as dimethyl sulfoxide is also added to improve fusion efficiency.

The ratio of immune cells to myeloma cells may be determined at one'sown discretion, preferably, for example, one myeloma cell for every oneto ten immunocytes. Culture media to be used for cell fusions include,for example, media that are suitable for the growth of myeloma celllines, such as RPMI1640 medium and MEM medium, and other conventionalculture medium used for this type of cell culture. In addition, serumsupplements such as fetal calf serum (FCS) may be preferably added tothe culture medium.

For cell fusion, predetermined amounts of the above immune cells andmyeloma cells are mixed well in the above culture medium. Then, a PEGsolution (for example, the average molecular weight is about 1,000 to6,000) prewarmed to about 37° C. is added thereto at a concentration ofgenerally 30% to 60% (w/v). This is gently mixed to produce desiredfusion cells (hybridomas). Then, an appropriate culture medium mentionedabove is gradually added to the cells, and this is repeatedlycentrifuged to remove the supernatant. Thus, cell fusion agents and suchwhich are unfavorable to hybridoma growth can be removed.

The hybridomas thus obtained can be selected by culture using aconventional selective medium, for example, HAT medium (a culture mediumcontaining hypoxanthine, aminopterin, and thymidine). Cells other thanthe desired hybridomas (non-fused cells) can be killed by continuingculture in the above HAT medium for a sufficient period of time.Typically, the period is several days to several weeks. Then, hybridomasproducing the desired antibody are screened and singly cloned byconventional limiting dilution methods.

The hybridomas thus obtained can be selected using a selection mediumbased on the selection marker possessed by the myeloma used for cellfusion. For example, HGPRT- or TK-deficient cells can be selected byculture using the HAT medium (a culture medium containing hypoxanthine,aminopterin, and thymidine). Specifically, when HAT-sensitive myelomacells are used for cell fusion, cells successfully fused with normalcells can selectively proliferate in the HAT medium. Cells other thanthe desired hybridomas (non-fused cells) can be killed by continuingculture in the above HAT medium for a sufficient period of time.Specifically, desired hybridomas can be selected by culture forgenerally several days to several weeks. Then, hybridomas producing thedesired antibody are screened and singly cloned by conventional limitingdilution methods.

Desired antibodies can be preferably selected and singly cloned byscreening methods based on known antigen/antibody reaction. For example,an IL-6 receptor-binding monoclonal antibody can bind to IL-6 receptorexpressed on the cell surface. Such a monoclonal antibody can bescreened by fluorescence activated cell sorting (FACS). FACS is a systemthat assesses the binding of an antibody to cell surface by analyzingcells contacted with a fluorescent antibody using laser beam, andmeasuring the fluorescence emitted from individual cells.

To screen for hybridomas that produce a monoclonal antibody of thepresent invention by FACS, IL-6 receptor-expressing cells are firstprepared. Cells preferably used for screening are mammalian cells inwhich IL-6 receptor is forcedly expressed. As control, the activity ofan antibody to bind to cell-surface IL-6 receptor can be selectivelydetected using non-transformed mammalian cells as host cells.Specifically, hybridomas producing an anti-IL-6 receptor monoclonalantibody can be isolated by selecting hybridomas that produce anantibody which binds to cells forced to express IL-6 receptor, but notto host cells.

Alternatively, the activity of an antibody to bind to immobilized IL-6receptor-expressing cells can be assessed based on the principle ofELISA. For example, IL-6 receptor-expressing cells are immobilized tothe wells of an ELISA plate. Culture supernatants of hybridomas arecontacted with the immobilized cells in the wells, and antibodies thatbind to the immobilized cells are detected. When the monoclonalantibodies are derived from mouse, antibodies bound to the cells can bedetected using an anti-mouse immunoglobulin antibody. Hybridomasproducing a desired antibody having the antigen-binding ability areselected by the above screening, and they can be cloned by a limitingdilution method or the like.

Monoclonal antibody-producing hybridomas thus prepared can be passagedin a conventional culture medium, and stored in liquid nitrogen for along period.

The above hybridomas are cultured by a conventional method, and desiredmonoclonal antibodies can be prepared from the culture supernatants.Alternatively, the hybridomas are administered to and grown incompatible mammals, and monoclonal antibodies are prepared from theascites. The former method is suitable for preparing antibodies withhigh purity.

Antibodies encoded by antibody genes that are cloned fromantibody-producing cells such as the above hybridomas can also bepreferably used. A cloned antibody gene is inserted into an appropriatevector, and this is introduced into a host to express the antibodyencoded by the gene. Methods for isolating antibody genes, inserting thegenes into vectors, and transforming host cells have already beenestablished, for example, by Vandamme et al. (Eur. J. Biochem. (1990)192(3), 767-775). Methods for producing recombinant antibodies are alsoknown as described below.

For example, a cDNA encoding the variable region (V region) of ananti-IL-6 receptor antibody is prepared from hybridoma cells expressingthe anti-IL-6 receptor antibody. For this purpose, total RNA is firstextracted from hybridomas. Methods used for extracting mRNAs from cellsinclude, for example:

the guanidine ultracentrifugation method (Biochemistry (1979) 18(24),5294-5299), and

the AGPC method (Anal. Biochem. (1987) 162(1), 156-159)

Extracted mRNAs can be purified using the mRNA Purification Kit (GEHealthcare Bioscience) or such. Alternatively, kits for extracting totalmRNA directly from cells, such as the QuickPrep mRNA Purification Kit(GE Healthcare Bioscience), are also commercially available. mRNAs canbe prepared from hybridomas using such kits. cDNAs encoding the antibodyV region can be synthesized from the prepared mRNAs using a reversetranscriptase.

cDNAs can be synthesized using the AMV Reverse TranscriptaseFirst-strand cDNA Synthesis Kit (Seikagaku Co.) or such. Furthermore,the SMART RACE cDNA amplification kit (Clontech) and the PCR-based5′-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002;Nucleic Acids Res. (1989) 17(8), 2919-2932) can be appropriately used tosynthesize and amplify cDNAs. In such a cDNA synthesis process,appropriate restriction enzyme sites described below may be introducedinto both ends of a cDNA.

The cDNA fragment of interest is purified from the resulting PCRproduct, and then this is ligated to a vector DNA. A recombinant vectoris thus constructed, and introduced into E. coli or such. After colonyselection, the desired recombinant vector can be prepared from thecolony-forming E. coli. Then, whether the recombinant vector has thecDNA nucleotide sequence of interest is tested by a known method such asthe dideoxy nucleotide chain termination method.

The 5′-RACE method which uses primers to amplify the variable regiongene is conveniently used for isolating the gene encoding the variableregion. First, a 5′-RACE cDNA library is constructed by cDNA synthesisusing RNAs extracted from hybridoma cells as a template. A commerciallyavailable kit such as the SMART RACE cDNA amplification kit isappropriately used to synthesize the 5′-RACE cDNA library.

The antibody gene is amplified by PCR using the prepared 5′-RACE cDNAlibrary as a template. Primers for amplifying the mouse antibody genecan be designed based on known antibody gene sequences. The nucleotidesequences of the primers vary depending on the immunoglobulin subclass.Therefore, it is preferable that the subclass is determined in advanceusing a commercially available kit such as the Iso Strip mousemonoclonal antibody isotyping kit (Roche Diagnostics).

Specifically, for example, primers that allow amplification of genesencoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains areused to isolate mouse IgG-encoding genes. In general, a primer thatanneals to a constant region site close to the variable region is usedas a 3′-side primer to amplify an IgG variable region gene. Meanwhile, aprimer attached to a 5′ RACE cDNA library construction kit is used as a5′-side primer.

PCR products thus amplified are used to reshape immunoglobulins composedof a combination of heavy and light chains. A desired antibody can beselected using the IL-6 receptor-binding activity of a reshapedimmunoglobulin as an indicator. For example, when the objective is toisolate an antibody against IL-6 receptor, it is more preferred that thebinding of the antibody to IL-6 receptor is specific. An IL-6receptor-binding antibody can be screened, for example, by the followingsteps:

(1) contacting an IL-6 receptor-expressing cell with an antibodycomprising the V region encoded by a cDNA isolated from a hybridoma;

(2) detecting the binding of the antibody to the IL-6receptor-expressing cell; and

(3) selecting an antibody that binds to the IL-6 receptor-expressingcell.

Methods for detecting the binding of an antibody to IL-6receptor-expressing cells are known. Specifically, the binding of anantibody to IL-6 receptor-expressing cells can be detected by theabove-described techniques such as FACS. Immobilized samples of IL-6receptor-expressing cells are appropriately used to assess the bindingactivity of an antibody.

Preferred antibody screening methods that use the binding activity as anindicator also include panning methods using phage vectors. Screeningmethods using phage vectors are advantageous when the antibody genes areisolated from heavy-chain and light-chain subclass libraries from apolyclonal antibody-expressing cell population. Genes encoding theheavy-chain and light-chain variable regions can be linked by anappropriate linker sequence to form a single-chain Fv (scFv). Phagespresenting scFv on their surface can be produced by inserting a geneencoding scFv into a phage vector. The phages are contacted with anantigen of interest. Then, a DNA encoding scFv having the bindingactivity of interest can be isolated by collecting phages bound to theantigen. This process can be repeated as necessary to enrich scFv havingthe binding activity of interest.

After isolation of the cDNA encoding the V region of the anti-IL-6receptor antibody of interest, the cDNA is digested with restrictionenzymes that recognize the restriction sites introduced into both endsof the cDNA. Preferred restriction enzymes recognize and cleave anucleotide sequence that occurs in the nucleotide sequence of theantibody gene at a low frequency. Furthermore, a restriction site for anenzyme that produces a sticky end is preferably introduced into a vectorto insert a single-copy digested fragment in the correct orientation.The cDNA encoding the V region of the anti-IL-6 receptor antibody isdigested as described above, and this is inserted into an appropriateexpression vector to construct an antibody expression vector. In thiscase, if a gene encoding the antibody constant region (C region) and agene encoding the above V region are fused in-frame, a chimeric antibodyis obtained. Herein, “chimeric antibody” means that the origin of theconstant region is different from that of the variable region. Thus, inaddition to mouse/human heterochimeric antibodies, human/humanallochimeric antibodies are included in the chimeric antibodies of thepresent invention. A chimeric antibody expression vector can beconstructed by inserting the above V region gene into an expressionvector that already has the constant region. Specifically, for example,a recognition sequence for a restriction enzyme that excises the above Vregion gene can be appropriately placed on the 5′ side of an expressionvector carrying a DNA encoding a desired antibody constant region (Cregion). A chimeric antibody expression vector is constructed by fusingin frame the two genes digested with the same combination of restrictionenzymes.

To produce an anti-IL-6 receptor monoclonal antibody, antibody genes areinserted into an expression vector so that the genes are expressed underthe control of an expression regulatory region. The expressionregulatory region for antibody expression includes, for example,enhancers and promoters. Furthermore, an appropriate signal sequence maybe attached to the amino terminus so that the expressed antibody issecreted to the outside of cells. In the Examples described later, apeptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO:3) are used as a signal sequence. Meanwhile, other appropriate signalsequences may be attached. The expressed polypeptide is cleaved at thecarboxyl terminus of the above sequence, and the resulting polypeptideis secreted to the outside of cells as a mature polypeptide. Then,appropriate host cells are transformed with the expression vector, andrecombinant cells expressing the anti-IL-6 receptor antibody-encoding

DNA are obtained.

DNAs encoding the antibody heavy chain (H chain) and light chain (Lchain) are separately inserted into different expression vectors toexpress the antibody gene. An antibody molecule having the H and Lchains can be expressed by co-transfecting the same host cell withvectors into which the H-chain and L-chain genes are respectivelyinserted. Alternatively, host cells can be transformed with a singleexpression vector into which DNAs encoding the H and L chains areinserted (see WO 1994/11523).

There are various known host cell/expression vector combinations forantibody preparation by introducing isolated antibody genes intoappropriate hosts. All of these expression systems are applicable toisolation of the antigen-binding domains of the present invention.Appropriate eukaryotic cells used as host cells include animal cells,plant cells, and fungal cells. Specifically, the animal cells include,for example, the following cells.

(1) mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK), HeLa,Vero, human embryonic kidney (HEK) 293, or such;(2) amphibian cells: Xenopus oocytes, or such; and(3) insect cells: sf9, sf21, Tn5, or such.

In addition, as a plant cell, an antibody gene expression system usingcells derived from the Nicotiana genus such as Nicotiana tabacum isknown. Callus cultured cells can be appropriately used to transformplant cells.

Furthermore, the following cells can be used as fungal cells:

-   -   yeasts: the Saccharomyces genus such as Saccharomyces        cerevisiae, and the Pichia genus such as Pichia pastoris; and    -   filamentous fungi: the Aspergillus genus such as Aspergillus        niger.

Furthermore, antibody gene expression systems that utilize prokaryoticcells are also known. For example, when using bacterial cells, E. colicells, Bacillus subtilis cells, and such can suitably be utilized in thepresent invention. Expression vectors carrying the antibody genes ofinterest are introduced into these cells by transfection. Thetransfected cells are cultured in vitro, and the desired antibody can beprepared from the culture of transformed cells.

In addition to the above-described host cells, transgenic animals canalso be used to produce a recombinant antibody. That is, the antibodycan be obtained from an animal into which the gene encoding the antibodyof interest is introduced. For example, the antibody gene can beconstructed as a fusion gene by inserting in frame into a gene thatencodes a protein produced specifically in milk. Goat (3-casein or suchcan be used, for example, as the protein secreted in milk. DNA fragmentscontaining the fused gene inserted with the antibody gene is injectedinto a goat embryo, and then this embryo is introduced into a femalegoat. Desired antibodies can be obtained as a protein fused with themilk protein from milk produced by the transgenic goat born from theembryo-recipient goat (or progeny thereof). In addition, to increase thevolume of milk containing the desired antibody produced by thetransgenic goat, hormones can be administered to the transgenic goat asnecessary (Ebert, K. M. et al., Bio/Technology (1994) 12 (7), 699-702).

When a polypeptide complex described herein is administered to human, anantigen-binding domain derived from a genetically recombinant antibodythat has been artificially modified to reduce the heterologousantigenicity against human and such, can be appropriately used as theantigen-binding domain of the complex. Such genetically recombinantantibodies include, for example, humanized antibodies. These modifiedantibodies are appropriately produced by known methods.

An antibody variable region used to produce the antigen-binding domainof a polypeptide complex described herein is generally formed by threecomplementarity-determining regions (CDRs) that are separated by fourframework regions (FRs). CDR is a region that substantially determinesthe binding specificity of an antibody. The amino acid sequences of CDRsare highly diverse. On the other hand, the FR-forming amino acidsequences often have high identity even among antibodies with differentbinding specificities. Therefore, generally, the binding specificity ofa certain antibody can be introduced to another antibody by CDRgrafting.

A humanized antibody is also called a reshaped human antibody.Specifically, humanized antibodies prepared by grafting the CDR of anon-human animal antibody such as a mouse antibody to a human antibodyand such are known. Common genetic engineering techniques for obtaininghumanized antibodies are also known. Specifically, for example, overlapextension PCR is known as a method for grafting a mouse antibody CDR toa human FR. In overlap extension PCR, a nucleotide sequence encoding amouse antibody CDR to be grafted is added to primers for synthesizing ahuman antibody FR. Primers are prepared for each of the four FRs. It isgenerally considered that when grafting a mouse CDR to a human FR,selecting a human FR that has high identity to a mouse FR isadvantageous for maintaining the CDR function. That is, it is generallypreferable to use a human FR comprising an amino acid sequence which hashigh identity to the amino acid sequence of the FR adjacent to the mouseCDR to be grafted.

Nucleotide sequences to be ligated are designed so that they will beconnected to each other in frame. Human FRs are individually synthesizedusing the respective primers. As a result, products in which the mouseCDR-encoding DNA is attached to the individual FR-encoding DNAs areobtained. Nucleotide sequences encoding the mouse CDR of each productare designed so that they overlap with each other. Then, complementarystrand synthesis reaction is conducted to anneal the overlapping CDRregions of the products synthesized using a human antibody gene astemplate. Human FRs are ligated via the mouse CDR sequences by thisreaction.

The full length V region gene, in which three CDRs and four FRs areultimately ligated, is amplified using primers that anneal to its 5′- or3′-end, which are added with suitable restriction enzyme recognitionsequences. An expression vector for humanized antibody can be producedby inserting the DNA obtained as described above and a DNA that encodesa human antibody C region into an expression vector so that they willligate in frame. After the recombinant vector is transfected into a hostto establish recombinant cells, the recombinant cells are cultured, andthe DNA encoding the humanized antibody is expressed to produce thehumanized antibody in the cell culture (see, European Patent PublicationNo. EP 239400 and

International Patent Publication No. WO 1996/002576).

By qualitatively or quantitatively measuring and evaluating theantigen-binding activity of the humanized antibody produced as describedabove, one can suitably select human antibody FRs that allow CDRs toform a favorable antigen-binding site when ligated through the CDRs.Amino acid residues in FRs may be substituted as necessary, so that theCDRs of a reshaped human antibody form an appropriate antigen-bindingsite. For example, amino acid sequence mutations can be introduced intoFRs by applying the PCR method used for grafting a mouse CDR into ahuman FR. More specifically, partial nucleotide sequence mutations canbe introduced into primers that anneal to the FR. Nucleotide sequencemutations are introduced into the FRs synthesized by using such primers.Mutant FR sequences having the desired characteristics can be selectedby measuring and evaluating the activity of the amino acid-substitutedmutant antibody to bind to the antigen by the above-mentioned method(Cancer Res. (1993) 53: 851-856).

Alternatively, desired human antibodies can be obtained by immunizingtransgenic animals having the entire repertoire of human antibody genes(see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO1996/034096; WO 1996/033735) by DNA immunization.

Furthermore, techniques for preparing human antibodies by panning usinghuman antibody libraries are also known. For example, the V region of ahuman antibody is expressed as a single-chain antibody (scFv) on phagesurface by the phage display method. Phages expressing an scFv thatbinds to the antigen can be selected. The DNA sequence encoding thehuman antibody V region that binds to the antigen can be determined byanalyzing the genes of selected phages. The DNA sequence of the scFvthat binds to the antigen is determined. An expression vector isprepared by fusing the V region sequence in frame with the C regionsequence of a desired human antibody, and inserting this into anappropriate expression vector. The expression vector is introduced intocells appropriate for expression such as those described above. Thehuman antibody can be produced by expressing the human antibody-encodinggene in the cells. These methods are already known (see WO 1992/001047;WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO1995/001438; WO 1995/015388).

In addition to the techniques described above, techniques of B cellcloning (identification of each antibody-encoding sequence, cloning andits isolation; use in constructing expression vector in order to prepareeach antibody (IgG1, IgG2, IgG3, or IgG4 in particular); and such) suchas described in Bernasconi et al. (Science (2002) 298: 2199-2202) or inWO 2008/081008 can be appropriately used to isolate antibody genes.

EU Numbering System and Kabat's Numbering System

According to the methods used in the present invention, amino acidpositions assigned to antibody CDR and FR are specified according toKabat's numbering (Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md., 1987 and 1991)). Herein,when an antigen-binding molecule is an antibody or antigen-bindingfragment, variable region amino acids are indicated according to Kabat'snumbering system, while constant region amino acids are indicatedaccording to EU numbering system based on Kabat's amino acid positions.

Conditions of Ion Concentration Conditions of Metal Ion Concentration

In a non-limiting embodiment of the present invention, the ionconcentration refers to a metal ion concentration. “Metal ions” refer toions of group I elements except hydrogen such as alkaline metals andcopper group elements, group II elements such as alkaline earth metalsand zinc group elements, group III elements except boron, group IVelements except carbon and silicon, group VIII elements such as irongroup and platinum group elements, elements belonging to subgroup A ofgroups V, VI, and VII, and metal elements such as antimony, bismuth, andpolonium. Metal atoms have the property of releasing valence electronsto become cations. This is referred to as ionization tendency. Metalswith strong ionization tendency are deemed to be chemically active.

In the present invention, preferred metal ions include, for example,calcium ion. Calcium ion is involved in modulation of many biologicalphenomena, including contraction of muscles such as skeletal, smooth,and cardiac muscles; activation of movement, phagocytosis, and the likeof leukocytes; activation of shape change, secretion, and the like ofplatelets; activation of lymphocytes; activation of mast cells includingsecretion of histamine; cell responses mediated by catecholamine areceptor or acetylcholine receptor; exocytosis; release of transmittersubstances from neuron terminals; and axoplasmic flow in neurons. Knownintracellular calcium ion receptors include troponin C, calmodulin,parvalbumin, and myosin light chain, which have several calciumion-binding sites and are believed to be derived from a common origin interms of molecular evolution. There are also many known calcium-bindingmotifs. Such well-known motifs include, for example, cadherin domains,EF-hand of calmodulin, C2 domain of Protein kinase C, Gla domain ofblood coagulation protein Factor IX, C-type lectins ofacyaroglycoprotein receptor and mannose-binding receptor, A domains ofLDL receptors, annexin, thrombospondin type 3 domain, and EGF-likedomains.

In the present invention, when the metal ion is calcium ion, theconditions of calcium ion concentration include low calcium ionconcentrations and high calcium ion concentrations. “The bindingactivity varies depending on calcium ion concentrations” means that theantigen-binding activity of an antigen-binding molecule varies due tothe difference in the conditions between low and high calcium ionconcentrations. For example, the antigen-binding activity of anantigen-binding molecule may be higher at a high calcium ionconcentration than at a low calcium ion concentration. Alternatively,the antigen-binding activity of an antigen-binding molecule may behigher at a low calcium ion concentration than at a high calcium ionconcentration.

Herein, the high calcium ion concentration is not particularly limitedto a specific value; however, the concentration may preferably beselected between 100 μM and 10 mM. In another embodiment, theconcentration may be selected between 200 μM and 5 mM. In an alternativeembodiment, the concentration may be selected between 500 μM and 2.5 mM.In still another embodiment, the concentration may be selected between200 μM and 2 mM. Furthermore, the concentration may be selected between400 μM and 1.5 mM. In particular, a concentration selected between 500μM and 2.5 mM, which is close to the plasma (blood) concentration ofcalcium ion in vivo, is preferred.

Herein, the low calcium ion concentration is not particularly limited toa specific value; however, the concentration may preferably be selectedbetween 0.1 μM and 30 μM. In another embodiment, the concentration maybe selected between 0.2 μM and 20 μM. In still another embodiment, theconcentration may be selected between 0.5 μM and 10 μM. In analternative embodiment, the concentration may be selected between 1 μMand 5 μM. Furthermore, the concentration may be selected between 2 μMand 4 μM. In particular, a concentration selected between 1 μM and 5 μM,which is close to the concentration of ionized calcium in earlyendosomes in vivo, is preferred.

Herein, “the antigen-binding activity is lower at a low calcium ionconcentration than at a high calcium ion concentration” means that theantigen-binding activity of an antigen-binding molecule is weaker at acalcium ion concentration selected between 0.1 μM and 30 μM than at acalcium ion concentration selected between 100 μM and 10 mM. Preferably,it means that the antigen-binding activity of an antigen-bindingmolecule is weaker at a calcium ion concentration selected between 0.5μM and 10 μM than at a calcium ion concentration selected between 200 μMand 5 mM. It particularly preferably means that the antigen-bindingactivity at the calcium ion concentration in the early endosome in vivois weaker than that at the in vivo plasma calcium ion concentration; andspecifically, it means that the antigen-binding activity of anantigen-binding molecule is weaker at a calcium ion concentrationselected between 1 μM and 5 μM than at a calcium ion concentrationselected between 500 μM and 2.5 mM.

Whether the antigen-binding activity of an antigen-binding molecule ischanged depending on metal ion concentrations can be determined, forexample, by the use of known measurement methods such as those describedin the section “Binding Activity” above. For example, in order toconfirm that the antigen-binding activity of an antigen-binding moleculebecomes higher at a high calcium ion concentration than at a low calciumion concentration, the antigen-binding activity of the antigen-bindingmolecule at low and high calcium ion concentrations is compared.

In the present invention, the expression “the antigen-binding activityis lower at a low calcium ion concentration than at a high calcium ionconcentration” can also be expressed as “the antigen-binding activity ofan antigen-binding molecule is higher at a high calcium ionconcentration than at a low calcium ion concentration”. In the presentinvention, “the antigen-binding activity is lower at a low calcium ionconcentration than at a high calcium ion concentration” is sometimeswritten as “the antigen-binding ability is weaker at a low calcium ionconcentration than at a high calcium ion concentration”. Also, “theantigen-binding activity at a low calcium ion concentration is reducedto be lower than that at a high calcium ion concentration” may bewritten as “the antigen-binding ability at a low calcium ionconcentration is made weaker than that at a high calcium ionconcentration”.

When determining the antigen-binding activity, the conditions other thancalcium ion concentration can be appropriately selected by those skilledin the art, and are not particularly limited. For example, the activitycan be determined at 37° C. in HEPES buffer. For example,

Biacore (GE Healthcare) or such can be used for the determination. Whenthe antigen is a soluble antigen, the antigen-binding activity of anantigen-binding molecule can be assessed by flowing the antigen as ananalyte over a chip onto which the antigen-binding molecule isimmobilized. When the antigen is a membrane antigen, the bindingactivity of an antigen-binding molecule to the membrane antigen can beassessed by flowing the antigen-binding molecule as an analyte over achip onto which the antigen is immobilized.

As long as the antigen-binding activity of an antigen-binding moleculeof the present invention is weaker at a low calcium ion concentrationthan at a high calcium ion concentration, the ratio of theantigen-binding activity between low and high calcium ion concentrationsis not particularly limited. However, the ratio of the KD (dissociationconstant) of the antigen-binding molecule for an antigen at a lowcalcium ion concentration with respect to the KD at a high calcium ionconcentration, i.e. the value of KD (3 μM Ca)/KD (2 mM Ca), ispreferably 2 or more, more preferably 10 or more, and still morepreferably 40 or more. The upper limit of the KD (3 μM Ca)/KD (2 mM Ca)value is not particularly limited, and may be any value such as 400,1000, or 10000 as long as the molecule can be produced by techniquesknown to those skilled in the art. Furthermore, it may also be specifiedby the KD (Ca 3 μM)/KD (Ca 1.2 mM) value. Specifically, the KD (Ca 3μM)/KD (Ca 1.2 mM) value is 2 or greater, preferably the KD (Ca 3 μM)/KD(Ca 1.2 mM) value is 10 or greater, and more preferably the KD (Ca 3μM)/KD (Ca 1.2 mM) value is 40 or greater. The upper limit of the KD (Ca3 μM)/KD (Ca 1.2 mM) value is not particularly limited, and may be anyvalue such as 400, 1000, or 10000 as long as the molecule can beproduced by techniques known to those skilled in the art.

When the antigen is a soluble antigen, KD (dissociation constant) can beused to represent the antigen-binding activity. Meanwhile, when theantigen is a membrane antigen, apparent KD (apparent dissociationconstant) can be used to represent the activity. KD (dissociationconstant) and apparent KD (apparent dissociation constant) can bedetermined by methods known to those skilled in the art, for example,using Biacore (GE healthcare), Scatchard plot, or flow cytometer.

Alternatively, for example, the dissociation rate constant (kd) can alsobe preferably used as an index to represent the ratio of theantigen-binding activity of an antigen-binding molecule of the presentinvention between low and high calcium concentrations. When thedissociation rate constant (kd) is used instead of the dissociationconstant (KD) as an index to represent the binding activity ratio, theratio of the dissociation rate constant (kd) between low and highcalcium concentrations, i.e. the value of kd (low calciumconcentration)/kd (high calcium concentration), is preferably 2 or more,more preferably 5 or more, still more preferably 10 or more, and yetmore preferably 30 or more. The upper limit of the Kd (low calciumconcentration)/kd (high calcium concentration) value is not particularlylimited, and can be any value such as 50, 100, or 200 as long as themolecule can be produced by techniques known to those skilled in theart.

When the antigen is a soluble antigen, kd (dissociation rate constant)can be used to represent the antigen-binding activity. Meanwhile, whenthe antigen is a membrane antigen, apparent kd (apparent dissociationrate constant) can be used to represent the antigen-binding activity.The kd (dissociation rate constant) and apparent kd (apparentdissociation rate constant) can be determined by methods known to thoseskilled in the art, for example, using Biacore (GE healthcare) or flowcytometer. In the present invention, when the antigen-binding activityof an antigen-binding molecule is determined at different calcium ionconcentrations, it is preferable to use the same conditions except forthe calcium concentrations.

For example, an antigen-binding domain or antibody whose antigen-bindingactivity is lower at a low calcium ion concentration than at a highcalcium ion concentration, which is one embodiment of the presentinvention, can be obtained via screening of antigen-binding domains orantibodies including the steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain or antibody at a low calcium concentration;

(b) determining the antigen-binding activity of an antigen-bindingdomain or antibody at a high calcium concentration; and

(c) selecting an antigen-binding domain or antibody whoseantigen-binding activity is lower at a low calcium concentration than ata high calcium concentration.

Moreover, an antigen-binding domain or antibody whose antigen-bindingactivity is lower at a low calcium ion concentration than at a highcalcium ion concentration, which is one embodiment of the presentinvention, can be obtained via screening of antigen-binding domains orantibodies, or a library thereof, including the steps of:

(a) contacting an antigen with an antigen-binding domain or antibody, ora library thereof at a high calcium concentration;(b) incubating at a low calcium concentration an antigen-binding domainor antibody that has bound to the antigen in step (a); and(c) isolating an antigen-binding domain or antibody dissociated in step(b).

Furthermore, an antigen-binding domain or antibody whose antigen-bindingactivity is lower at a low calcium ion concentration than at a highcalcium ion concentration, which is one embodiment of the presentinvention, can be obtained via screening of antigen-binding domains orantibodies, or a library thereof, including the steps of:

(a) contacting an antigen with a library of antigen-binding domains orantibodies at a low calcium concentration;(b) selecting an antigen-binding domain or antibody which does not bindto the antigen in step (a);(c) allowing the antigen-binding domain or antibody selected in step (c)to bind to the antigen at a high calcium concentration; and(d) isolating an antigen-binding domain or antibody that has bound tothe antigen in step (c).

In addition, an antigen-binding domain or antibody whose antigen-bindingactivity is lower at a low calcium ion concentration than at a highcalcium ion concentration, which is one embodiment of the presentinvention, can be obtained by a screening method comprising the stepsof:

(a) contacting at a high calcium concentration a library ofantigen-binding domains or antibodies with a column onto which anantigen is immobilized;(b) eluting an antigen-binding domain or antibody that has bound to thecolumn in step (a) from the column at a low calcium concentration; and(c) isolating the antigen-binding domain or antibody eluted in step (b).

Furthermore, an antigen-binding domain or antibody whose antigen-bindingactivity is lower at a low calcium ion concentration than at a highcalcium ion concentration, which is one embodiment of the presentinvention, can be obtained by a screening method comprising the stepsof:

(a) allowing at a low calcium concentration a library of antigen-bindingdomains or antibodies to pass through a column onto which an antigen isimmobilized;(b) collecting an antigen-binding domain or antibody that has beeneluted without binding to the column in step (a);(c) allowing the antigen-binding domain or antibody collected in step(b) to bind to the antigen at a high calcium concentration; and(d) isolating an antigen-binding domain or antibody that has bound tothe antigen in step (c).

Moreover, an antigen-binding domain or antibody whose antigen-bindingactivity is lower at a low calcium ion concentration than at a highcalcium ion concentration, which is one embodiment of the presentinvention, can be obtained by a screening method comprising the stepsof:

(a) contacting an antigen with a library of antigen-binding domains orantibodies at a high calcium concentration;(b) obtaining an antigen-binding domain or antibody that has bound tothe antigen in step (a);(c) incubating at a low calcium concentration the antigen-binding domainor antibody obtained in step (b); and(d) isolating an antigen-binding domain or antibody whoseantigen-binding activity in step (c) is weaker than the criterion forthe selection of step (b).

The above-described steps may be repeated twice or more times. Thus, thepresent invention provides antigen-binding domains or antibodies whoseantigen-binding activity is lower at a low calcium ion concentrationthan at a high calcium ion concentration, which are obtained byscreening methods that further comprises the step of repeating twice ormore times steps (a) to (c) or (a) to (d) in the above-describedscreening methods. The number of cycles of steps (a) to (c) or (a) to(d) is not particularly limited, but generally is 10 or less.

In the screening methods of the present invention, the antigen-bindingactivity of an antigen-binding domain or antibody at a low calciumconcentration is not particularly limited as long as it isantigen-binding activity at an ionized calcium concentration of between0.1 μM and 30 μM, but preferably is antigen-binding activity at anionized calcium concentration of between 0.5 μM and 10 μM. Morepreferably, it is antigen-binding activity at the ionized calciumconcentration in the early endosome in vivo, specifically, between 1 μMand 5 μM. Meanwhile, the antigen-binding activity of an antigen-bindingdomain or antibody at a high calcium concentration is not particularlylimited, as long as it is antigen-binding activity at an ionized calciumconcentration of between 100 μM and 10 mM, but preferably isantigen-binding activity at an ionized calcium concentration of between200 μM and 5 mM. More preferably, it is antigen-binding activity at theionized calcium concentration in plasma in vivo, specifically, between0.5 mM and 2.5 mM.

The antigen-binding activity of an antigen-binding domain or antibodycan be measured by methods known to those skilled in the art. Conditionsother than the ionized calcium concentration can be determined by thoseskilled in the art. The antigen-binding activity of an antigen-bindingdomain or antibody can be evaluated as a dissociation constant (KD),apparent dissociation constant (apparent KD), dissociation rate constant(kd), apparent dissociation constant (apparent kd), and such. These canbe determined by methods known to those skilled in the art, for example,using Biacore (GE healthcare), Scatchard plot, or FACS.

In the present invention, the step of selecting an antigen-bindingdomain or antibody whose antigen-binding activity is higher at a highcalcium concentration than at a low calcium concentration is synonymouswith the step of selecting an antigen-binding domain or antibody whoseantigen-binding activity is lower at a low calcium concentration than ata high calcium concentration.

As long as the antigen-binding activity is higher at a high calciumconcentration than at a low calcium concentration, the difference in theantigen-binding activity between high and low calcium concentrations isnot particularly limited; however, the antigen-binding activity at ahigh calcium concentration is preferably twice or more, more preferably10 times or more, and still more preferably 40 times or more than thatat a low calcium concentration.

Antigen-binding domains or antibodies of the present invention to bescreened by the screening methods described above may be anyantigen-binding domains and antibodies. For example, it is possible toscreen the above-described antigen-binding domains or antibodies. Forexample, antigen-binding domains or antibodies having natural sequencesor substituted amino acid sequences may be screened.

Libraries

In an embodiment, an antigen-binding domain or antibody of the presentinvention can be obtained from a library that is mainly composed of aplurality of antigen-binding molecules whose sequences are differentfrom one another and whose antigen-binding domains have at least oneamino acid residue that alters the antigen-binding activity of theantigen-binding molecules depending on ion concentrations. The ionconcentrations preferably include, for example, metal ion concentrationand hydrogen ion concentration.

Herein, a “library” refers to a plurality of antigen-binding moleculesor a plurality of fusion polypeptides containing antigen-bindingmolecules, or nucleic acids or polynucleotides encoding their sequences.The sequences of a plurality of antigen-binding molecules or a pluralityof fusion polypeptides containing antigen-binding molecules in a libraryare not identical, but are different from one another.

Herein, the phrase “sequences are different from one another” in theexpression “a plurality of antigen-binding molecules whose sequences aredifferent from one another” means that the sequences of antigen-bindingmolecules in a library are different from one another. Specifically, ina library, the number of sequences different from one another reflectsthe number of independent clones with different sequences, and may alsobe referred to as “library size”. The library size of a conventionalphage display library ranges from 10⁶ to 10¹². The library size can beincreased up to 10¹⁴ by the use of known techniques such as ribosomedisplay. However, the actual number of phage particles used in panningselection of a phage library is in general 10-10000 times greater thanthe library size. This excess multiplicity is also referred to as “thenumber of library equivalents”, and means that there are 10 to 10,000individual clones that have the same amino acid sequence. Thus, in thepresent invention, the phrase “sequences are different from one another”means that the sequences of independent antigen-binding molecules in alibrary, excluding library equivalents, are different from one another.More specifically, the above means that there are 10⁶ to 10¹⁴antigen-binding molecules whose sequences are different from oneanother, preferably 10⁷ to 10¹² molecules, more preferably 10⁸ to 10¹¹molecules, and particularly preferably 10⁸ to 10¹² molecules whosesequences are different from one another.

Herein, the phrase “a plurality of” in the expression “a library mainlycomposed of a plurality of antigen-binding molecules” generally refersto, in the case of, for example, antigen-binding molecules, fusionpolypeptides, polynucleotide molecules, vectors, or viruses of thepresent invention, a group of two or more types of the substance. Forexample, when two or more substances are different from one another in aparticular characteristic, this means that there are two or more typesof the substance. Such examples may include, for example, mutant aminoacids observed at specific amino acid positions in an amino acidsequence. For example, when there are two or more antigen-bindingmolecules of the present invention whose sequences are substantially thesame or preferably the same except for flexible residues or except forparticular mutant amino acids at hypervariable positions exposed on thesurface, there are a plurality of antigen-binding molecules of thepresent invention. In another example, when there are two or morepolynucleotide molecules whose sequences are substantially the same orpreferably the same except for nucleotides encoding flexible residues ornucleotides encoding mutant amino acids of hypervariable positionsexposed on the surface, there are a plurality of polynucleotidemolecules of the present invention.

In addition, herein, the phrase “mainly composed of” in the expression“a library mainly composed of a plurality of antigen-binding molecules”reflects the number of antigen-binding molecules whose antigen-bindingactivity varies depending on ion concentrations, among independentclones with different sequences in a library. Specifically, it ispreferable that there are at least 10⁴ antigen-binding molecules havingsuch binding activity in a library. More preferably, antigen-bindingdomains of the present invention can be obtained from a librarycontaining at least 10⁵ antigen-binding molecules having such bindingactivity. Still more preferably, antigen-binding domains of the presentinvention can be obtained from a library containing at least 10⁶antigen-binding molecules having such binding activity. Particularlypreferably, antigen-binding domains of the present invention can beobtained from a library containing at least 10′ antigen-bindingmolecules having such binding activity. Yet more preferably,antigen-binding domains of the present invention can be obtained from alibrary containing at least 10⁸ antigen-binding molecules having suchbinding activity. Alternatively, this may also be preferably expressedas the ratio of the number of antigen-binding molecules whoseantigen-binding activity varies depending on ion concentrations withrespect to the number of independent clones having different sequencesin a library. Specifically, antigen-binding domains of the presentinvention can be obtained from a library in which antigen-bindingmolecules having such binding activity account for 0.1% to 80%,preferably 0.5% to 60%, more preferably 1% to 40%, still more preferably2% to 20%, and particularly preferably 4% to 10% of independent cloneswith different sequences in the library. In the case of fusionpolypeptides, polynucleotide molecules, or vectors, similar expressionsmay be possible using the number of molecules or the ratio to the totalnumber of molecules. In the case of viruses, similar expressions mayalso be possible using the number of virions or the ratio to totalnumber of virions.

Amino Acids that Alter the Antigen-Binding Activity of Antigen-BindingDomains Depending on Calcium Ion Concentrations

Antigen-binding domains or antibodies of the present invention to bescreened by the above-described screening methods may be prepared in anymanner. For example, when the metal ion is calcium ion, it is possibleto use preexisting antibodies, preexisting libraries (phage library,etc.), antibodies or libraries prepared from hybridomas obtained byimmunizing animals or from B cells of immunized animals, antibodies orlibraries obtained by introducing amino acids capable of chelatingcalcium (for example, aspartic acid and glutamic acid) or unnaturalamino acid mutations into the above-described antibodies or libraries(calcium-chelatable amino acids (such as aspartic acid and glutamicacid), libraries with increased content of unnatural amino acids,libraries prepared by introducing calcium-chelatable amino acids (suchas aspartic acid and glutamic acid) or unnatural amino acid mutations atparticular positions, or the like.

Examples of the amino acids that alter the antigen-binding activity ofantigen-binding molecules depending on ion concentrations as describedabove may be any types of amino acids as long as the amino acids form acalcium-binding motif. Calcium-binding motifs are well known to thoseskilled in the art and have been described in details (for example,Springer et al. (Cell (2000) 102, 275-277); Kawasaki and Kretsinger(Protein Prof (1995) 2, 305-490); Moncrief et al. (J. Mol. Evol. (1990)30, 522-562); Chauvaux et al. (Biochem. J. (1990) 265, 261-265); Bairochand Cox (FEBS Lett. (1990) 269, 454-456); Davis (New Biol. (1990) 2,410-419); Schaefer et al. (Genomics (1995) 25, 638-643); Economou et al.(EMBO J. (1990) 9, 349-354); Wurzburg et al. (Structure. (2006) 14, 6,1049-1058)). Specifically, any known calcium-binding motifs, includingtype C lectins such as ASGPR, CD23, MBR, and DC-SIGN, can be included inantigen-binding molecules of the present invention. Preferred examplesof such preferred calcium-binding motifs also include, in addition tothose described above, for example, the calcium-binding motif in theantigen-binding domain of SEQ ID NO: 4, 9, or 10.

Furthermore, as amino acids that alter the antigen-binding activity ofantigen-binding molecules depending on calcium ion concentrations, forexample, amino acids having metal-chelating activity may also bepreferably used. Examples of such metal-chelating amino acids include,for example, serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)),glutamine

(Gln(Q)), aspartic acid (Asp(D)), and glutamic acid (Glu(E)).

Positions in the antigen-binding domains at which the above-describedamino acids are contained are not particularly limited to particularpositions, and may be any positions within the heavy chain variableregion or light chain variable region that forms an antigen-bindingdomain, as long as they alter the antigen-binding activity ofantigen-binding molecules depending on calcium ion concentrations.Specifically, antigen-binding domains of the present invention can beobtained from a library mainly composed of antigen-binding moleculeswhose sequences are different from one another and whose heavy chainantigen-binding domains contain amino acids that alter theantigen-binding activity of the antigen-binding molecules depending oncalcium ion concentrations. In another non-limiting embodiment,antigen-binding domains of the present invention can be obtained from alibrary mainly composed of antigen-binding molecules whose sequences aredifferent from one another and whose heavy chain CDR3 domains containthe above-mentioned amino acids. In still another non-limitingembodiment, antigen-binding domains of the present invention can beobtained from a library mainly composed of antigen-binding moleculeswhose sequences are different from one another and whose heavy chainCDR3 domains contain the above-mentioned amino acids at positions 95,96, 100a, and/or 101 as indicated according to the Kabat numberingsystem.

Meanwhile, in a non-limiting embodiment of the present invention,antigen-binding domains of the present invention can be obtained from alibrary mainly composed of antigen-binding molecules whose sequences aredifferent from one another and whose light chain antigen-binding domainscontain amino acids that alter the antigen-binding activity ofantigen-binding molecules depending on calcium ion concentrations. Inanother embodiment, antigen-binding domains of the present invention canbe obtained from a library mainly composed of antigen-binding moleculeswhose sequences are different from one another and whose light chainCDR1 domains contain the above-mentioned amino acids. In still anotherembodiment, antigen-binding domains of the present invention can beobtained from a library mainly composed of antigen-binding moleculeswhose sequences are different from one another and whose light chainCDR1 domains contain the above-mentioned amino acids at positions 30,31, and/or 32 as indicated according to the Kabat numbering system.

In another non-limiting embodiment, antigen-binding domains of thepresent invention can be obtained from a library mainly composed ofantigen-binding molecules whose sequences are different from one anotherand whose light chain CDR2 domains contain the above-mentioned aminoacid residues. In yet another embodiment, the present invention provideslibraries mainly composed of antigen-binding molecules whose sequencesare different from one another and whose light chain CDR2 domainscontain the above-mentioned amino acid residues at position 50 asindicated according to the Kabat numbering system.

In still another non-limiting embodiment of the present invention,antigen-binding domains of the present invention can be obtained from alibrary mainly composed of antigen-binding molecules whose sequences aredifferent from one another and whose light chain CDR3 domains containthe above-mentioned amino acid residues. In an alternative embodiment,antigen-binding domains of the present invention can be obtained from alibrary mainly composed of antigen-binding molecules whose sequences aredifferent from one another and whose light chain CDR3 domains containthe above-mentioned amino acid residues at position 92 as indicatedaccording to the Kabat numbering system.

Furthermore, in a different embodiment of the present invention,antigen-binding domains of the present invention can be obtained from alibrary mainly composed of antigen-binding molecules whose sequences aredifferent from one another and in which two or three CDRs selected fromthe above-described light chain CDR1, CDR2, and CDR3 contain theaforementioned amino acid residues. Moreover, antigen-binding domains ofthe present invention can be obtained from a library mainly composed ofantigen-binding molecules whose sequences are different from one anotherand whose light chains contain the aforementioned amino acid residues atany one or more of positions 30, 31, 32, 50, and/or 92 as indicatedaccording to the Kabat numbering system.

In a particularly preferred embodiment, the framework sequences of thelight chain and/or heavy chain variable region of an antigen-bindingmolecule preferably contain human germ line framework sequences. Thus,in an embodiment of the present invention, when the framework sequencesare completely human sequences, it is expected that when such anantigen-binding molecule of the present invention is administered tohumans (for example, to treat diseases), it induces little or noimmunogenic response. In the above sense, the phrase “containing a germline sequence” in the present invention means that a part of theframework sequences of the present invention is identical to a part ofany human germ line framework sequences. For example, when the heavychain FR2 sequence of an antigen-binding molecule of the presentinvention is a combination of heavy chain FR2 sequences of differenthuman germ line framework sequences, such a molecule is also anantigen-binding molecule of the present invention “containing a germline sequence”.

Preferred examples of the frameworks include, for example, fully humanframework region sequences currently known, which are included in thewebsite of V-Base (http://vbase.mrc-cpe.cam.ac.uk/) or others. Thoseframework region sequences can be appropriately used as a germ linesequence contained in an antigen-binding molecule of the presentinvention. The germ line sequences may be categorized according to theirsimilarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798);Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461); Cox et al.(Nat. Genetics (1994) 7, 162-168)). Appropriate germ line sequences canbe selected from Vκ, which is grouped into seven subgroups; Vλ, which isgrouped into ten subgroups; and VH, which is grouped into sevensubgroups.

Fully human VH sequences preferably include, but are not limited to, forexample, VH sequences of:

subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45,VH1-46, VH1-58, and VH1-69);subgroup VH2 (for example, VH2-5, VH2-26, and VH2-70);subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20,VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49,VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, and VH3-74);subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, andVH4-61);subgroup VH5 (VH5-51);subgroup VH6 (VH6-1); andsubgroup VH7 (VH7-4 and VH7-81).

These are also described in known documents (Matsuda et al. (J. Exp.Med. (1998) 188, 1973-1975)) and such, and thus persons skilled in theart can appropriately design antigen-binding molecules of the presentinvention based on the information of these sequences. It is alsopreferable to use other fully human frameworks or framework sub-regions.

Fully human VK sequences preferably include, but are not limited to, forexample:

A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23,L24, O2, O4, O8, O12, O14, and O18 grouped into subgroup Vk1;A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, grouped intosubgroup Vk2;A11, A27, L2, L6, L10, L16, L20, and L25, grouped into subgroup Vk3;B3, grouped into subgroup Vk4;B2 (herein also referred to as Vk5-2), grouped into subgroup Vk5; andA10, A14, and A26, grouped into subgroup VK6(Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable andZachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022);Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

Fully human VL sequences preferably include, but are not limited to, forexample:

V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18,V1-19, V1-20, and V1-22, grouped into subgroup VL1;V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19,grouped into subgroup VL1;V3-2, V3-3, and V3-4, grouped into subgroup VL3;V4-1, V4-2, V4-3, V4-4, and V4-6, grouped into subgroup VL4; andV5-1, V5-2, V5-4, and V5-6, grouped into subgroup VL5 (Kawasaki et al.(Genome Res. (1997) 7, 250-261)).

Normally, these framework sequences are different from one another atone or more amino acid residues. These framework sequences can be usedin combination with “at least one amino acid residue that alters theantigen-binding activity of an antigen-binding molecule depending on ionconcentrations” of the present invention. Other examples of the fullyhuman frameworks used in combination with “at least one amino acidresidue that alters the antigen-binding activity of an antigen-bindingmolecule depending on ion concentrations” of the present inventioninclude, but are not limited to, for example, KOL, NEWM, REI, EU, TUR,TEI, LAY, and POM (for example, Kabat et al. (1991) supra; Wu et al. (J.Exp. Med. (1970) 132, 211-250)).

Without being bound by a particular theory, one reason for theexpectation that the use of germ line sequences precludes adverse immuneresponses in most individuals is believed to be as follows. As a resultof the process of affinity maturation during normal immune responses,somatic mutation occurs frequently in the variable regions ofimmunoglobulin. Such mutations mostly occur around CDRs whose sequencesare hypervariable, but also affect residues of framework regions. Suchframework mutations do not exist on the germ line genes, and also theyare less likely to be immunogenic in patients. On the other hand, thenormal human population is exposed to most of the framework sequencesexpressed from the germ line genes. As a result of immunotolerance,these germ line frameworks are expected to have low or no immunogenicityin patients. To maximize the possibility of immunotolerance, variableregion-encoding genes may be selected from a group of commonly occurringfunctional germ line genes.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc.Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR canbe appropriately employed to produce antigen-binding molecules of thepresent invention in which the above-described framework sequencescontain amino acids that alter the antigen-binding activity of theantigen-binding molecules depending on calcium ion concentrations.

For example, a library which contains a plurality of antigen-bindingmolecules of the present invention whose sequences are different fromone another can be constructed by combining heavy chain variable regionsprepared as a randomized variable region sequence library with a lightchain variable region selected as a framework sequence originallycontaining at least one amino acid residue that alters theantigen-binding activity of the antigen-binding molecule depending oncalcium ion concentrations. Non-limiting examples of such libraries whenthe ion concentration is calcium ion concentration include a library inwhich a light chain variable region sequence belonging to the Vk5-2family represented by the light chain variable region sequence of SEQ IDNO: 4 (Vk5-2) is combined with heavy chain variable regions produced asa randomized variable region sequence library.

Alternatively, a light chain variable region sequence selected as aframework region originally containing at least one amino acid residuethat alters the antigen-binding activity of an antigen-binding moleculeas mentioned above can be design to contain various amino acid residuesother than the above amino acid residues. Herein, such residues arereferred to as flexible residues. The number and position of flexibleresidues are not particularly limited as long as the antigen-bindingactivity of the antigen-binding molecule of the present invention variesdepending on ion concentrations. Specifically, the CDR sequences and/orFR sequences of the heavy chain and/or light chain may contain one ormore flexible residues. For example, when the ion concentration iscalcium ion concentration, non-limiting examples of flexible residues tobe introduced into the light chain variable region sequence of SEQ IDNO: 4 (Vk5-2) include the amino acid residues listed in Tables 1 or 2.

TABLE 1 Kabat CDR numbering Amino acid in 70% of the total CDR1 28 S:100% 29 I: 100% 30 E: 72% N: 14% S: 14% 31 D: 100% 32 D: 100% 33 L: 100%34 A: 70% N: 30% CDR2 50 E: 100% 51 A: 100% 52 S: 100% 53 H: 5% N: 25%S: 45% T: 25% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 25%S: 15% R: 15% Y: 45% 92 D: 80% N: 10% S: 10% 93 D: 5% G: 10% N: 25% S:50% R: 10% 94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50%

TABLE 2 Kabat CDR numbering Amino acid in 30% of the total CDR1 28 S:100% 29 I: 100% 30 E: 83% S: 17% 31 D: 100% 32 D: 100% 33 L: 100% 34 A:70% N: 30% CDR2 50 H: 100% 51 A: 100% 52 S: 100% 53 H: 5% N: 25% S: 45%T: 25% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 25% S: 15%R: 15% Y: 45% 92 D: 80% N: 10% S: 10% 93 D: 5% G: 10% N: 25% S: 50% R:10% 94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50%

Herein, flexible residues refer to amino acid residue variations presentat hypervariable positions at which several different amino acids arepresent on the light chain and heavy chain variable regions when theamino acid sequences of known and/or native antibodies orantigen-binding domains are compared. Hypervariable positions aregenerally located in the CDR regions. In an embodiment, the dataprovided by Kabat, Sequences of Proteins of Immunological Interest(National Institute of Health Bethesda Md.) (1987 and 1991) is useful todetermine hypervariable positions in known and/or native antibodies.Furthermore, databases on the Internet (http://vbase.mrc-cpe.cam.ac.uk/,http://www.bioinforg.uk/abs/index.html) provide the collected sequencesof many human light chains and heavy chains and their locations. Theinformation on the sequences and locations is useful to determinehypervariable positions in the present invention. According to thepresent invention, when a certain amino acid position has preferablyabout 2 to about 20 possible amino acid residue variations, preferablyabout 3 to about 19, preferably about 4 to about 18, preferably 5 to 17,preferably 6 to 16, preferably 7 to 15, preferably 8 to 14, preferably 9to 13, and preferably 10 to 12 possible amino acid residue variations,the position is hypervariable. In some embodiments, a certain amino acidposition may have preferably at least about 2, preferably at least about4, preferably at least about 6, preferably at least about 8, preferablyabout 10, and preferably about 12 amino acid residue variations.

Alternatively, a library containing a plurality of antigen-bindingmolecules of the present invention whose sequences are different fromone another can be constructed by combining heavy chain variable regionsproduced as a randomized variable region sequence library with lightchain variable regions into which at least one amino acid residue thatalters the antigen-binding activity of antigen-binding moleculesdepending on ion concentrations as mentioned above is introduced. Whenthe ion concentration is calcium ion concentration, non-limitingexamples of such libraries preferably include, for example, libraries inwhich heavy chain variable regions produced as a randomized variableregion sequence library are combined with light chain variable regionsequences in which a particular residue(s) in a germ line sequence suchas SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), or SEQ IDNO: 8 (Vk4) has been substituted with at least one amino acid residuethat alters the antigen-binding activity of an antigen-binding moleculedepending on calcium ion concentrations. Non-limiting examples of suchamino acid residues include amino acid residues in light chain CDR1.Furthermore, non-limiting examples of such amino acid residues includeamino acid residues in light chain CDR2. In addition, non-limitingexamples of such amino acid residues also include amino acid residues inlight chain CDR3.

Non-limiting examples of such amino acid residues contained in lightchain CDR1 include those at positions 30, 31, and/or 32 in the CDR1 oflight chain variable region as indicated by Kabat numbering.Furthermore, non-limiting examples of such amino acid residues containedin light chain CDR2 include an amino acid residue at position 50 in theCDR2 of light chain variable region as indicated by Kabat numbering.Moreover, non-limiting examples of such amino acid residues contained inlight chain CDR3 include an amino acid residue at position 92 in theCDR3 of light chain variable region as indicated by Kabat numbering.These amino acid residues can be contained alone or in combination aslong as they form a calcium-binding motif and/or as long as theantigen-binding activity of an antigen-binding molecule varies dependingon calcium ion concentrations. Meanwhile, as troponin C, calmodulin,parvalbumin, and myosin light chain, which have several calciumion-binding sites and are believed to be derived from a common origin interms of molecular evolution, are known, the light chain CDR1, CDR2,and/or CDR3 can be designed to have their binding motifs. For example,it is possible to use cadherin domains, EF hand of calmodulin, C2 domainof Protein kinase C, Gla domain of blood coagulation protein FactorIX, Ctype lectins of acyaroglycoprotein receptor and mannose-bindingreceptor, A domains of LDL receptors, annexin, thrombospondin type 3domain, and EGF-like domains in an appropriate manner for the abovepurposes.

When heavy chain variable regions produced as a randomized variableregion sequence library and light chain variable regions into which atleast one amino acid residue that alters the antigen-binding activity ofan antigen-binding molecule depending on ion concentrations has beenintroduced are combined as described above, the sequences of the lightchain variable regions can be designed to contain flexible residues inthe same manner as described above. The number and position of suchflexible residues are not particularly limited to particular embodimentsas long as the antigen-binding activity of antigen-binding molecules ofthe present invention varies depending on ion concentrations.Specifically, the CDR sequences and/or FR sequences of heavy chainand/or light chain can contain one or more flexible residues. When theion concentration is calcium ion concentration, non-limiting examples offlexible residues to be introduced into the sequence of light chainvariable region include the amino acid residues listed in Tables 1 and2.

The preferred heavy chain variable regions to be combined include, forexample, randomized variable region libraries. Known methods arecombined as appropriate to produce a randomized variable region library.In a non-limiting embodiment of the present invention, an immune libraryconstructed based on antibody genes derived from lymphocytes of animalsimmunized with a specific antigen, patients with infections, personswith an elevated antibody titer in blood as a result of vaccination,cancer patients, or auto immune disease patients, may be preferably usedas a randomized variable region library.

In another non-limiting embodiment of the present invention, a syntheticlibrary produced by replacing the CDR sequences of V genes in genomicDNA or functional reshaped V genes with a set of syntheticoligonucleotides containing sequences encoding codon sets of anappropriate length can also be preferably used as a randomized variableregion library. In this case, since sequence diversity is observed inthe heavy chain CDR3 sequence, it is also possible to replace the CDR3sequence only. A criterion of giving rise to diversity in amino acids inthe variable region of an antigen-binding molecule is that diversity isgiven to amino acid residues at surface-exposed positions in theantigen-binding molecule. The surface-exposed position refers to aposition that is considered to be able to be exposed on the surfaceand/or contacted with an antigen, based on structure, ensemble ofstructures, and/or modeled structure of an antigen-binding molecule. Ingeneral, such positions are CDRs. Preferably, surface-exposed positionsare determined using coordinates from a three-dimensional model of anantigen-binding molecule using a computer program such as the InsightIIprogram (Accelrys). Surface-exposed positions can be determined usingalgorithms known in the art (for example, Lee and Richards (J. Mol.Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16,548-558)). Determination of surface-exposed positions can be performedusing software suitable for protein modeling and three-dimensionalstructural information obtained from an antibody. Software that can beused for these purposes preferably includes SYBYL Biopolymer Modulesoftware (Tripos Associates). Generally or preferably, when an algorithmrequires a user input size parameter, the “size” of a probe which isused in the calculation is set at about 1.4 Angstrom or smaller inradius. Furthermore, methods for determining surface-exposed regions andareas using software for personal computers are described by Pacios(Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46-53).

In another non-limiting embodiment of the present invention, a naivelibrary, which is constructed from antibody genes derived fromlymphocytes of healthy persons and whose repertoire consists of naivesequences, which are antibody sequences with no bias, can also beparticularly preferably used as a randomized variable region library(Gejima et al. (Human Antibodies (2002) 11, 121-129); Cardoso et al.(Scand. J. Immunol. (2000) 51, 337-344)). Herein, an amino acid sequencecomprising a naive sequence refers to an amino acid sequence obtainedfrom such a naive library.

In one embodiment of the present invention, an antigen-binding domain ofthe present invention can be obtained from a library containing aplurality of antigen-binding molecules of the present invention whosesequences are different from one another, prepared by combining lightchain variable regions constructed as a randomized variable regionsequence library with a heavy chain variable region selected as aframework sequence that originally contains “at least one amino acidresidue that alters the antigen-binding activity of an antigen-bindingmolecule depending on ion concentrations”. When the ion concentration iscalcium ion concentration, non-limiting examples of such librariespreferably include those constructed by combining light chain variableregions constructed as a randomized variable region sequence librarywith the sequence of heavy chain variable region of SEQ ID NO: 9(6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1). Alternatively, such alibrary can be constructed by selecting appropriate light chain variableregions from those having germ line sequences, instead of light chainvariable regions constructed as a randomized variable region sequencelibrary. Such preferred libraries include, for example, those in whichthe sequence of heavy chain variable region of SEQ ID NO: 9 (6RL#9-IgG1)or SEQ ID NO: 10 (6KC4-1#85-IgG1) is combined with light chain variableregions having germ line sequences.

Alternatively, the sequence of a heavy chain variable region selected asa framework sequence that originally contains “at least one amino acidresidue that alters the antigen-binding activity of an antigen-bindingmolecule” as mentioned above can be designed to contain flexibleresidues. The number and position of the flexible residues are notparticularly limited as long as the antigen-binding activity of anantigen-binding molecule of the present invention varies depending onion concentrations. Specifically, the CDR and/or FR sequences of heavychain and/or light chain can contain one or more flexible residues. Whenthe ion concentration is calcium ion concentration, non-limitingexamples of flexible residues to be introduced into the sequence ofheavy chain variable region of SEQ ID NO: 9 (6RL#9-IgG1) include allamino acid residues of heavy chain CDR1 and CDR2 and the amino acidresidues of the heavy chain CDR3 except those at positions 95, 96,and/or 100a as indicated by Kabat numbering. Alternatively, non-limitingexamples of flexible residues to be introduced into the sequence ofheavy chain variable region of SEQ ID NO: 10 (6KC4-1#85-IgG1) includeall amino acid residues of heavy chain CDR1 and CDR2 and the amino acidresidues of the heavy chain CDR3 except those at amino acid positions 95and/or 101 as indicated by Kabat numbering.

Alternatively, a library containing a plurality of antigen-bindingmolecules whose sequences are different from one another can beconstructed by combining light chain variable regions constructed as arandomized variable region sequence library or light chain variableregions having germ line sequences with heavy chain variable regionsinto which “at least one amino acid residue responsible for the ionconcentration-dependent change in the antigen-binding activity of anantigen-binding molecule” has been introduced as mentioned above. Whenthe ion concentration is calcium ion concentration, non-limitingexamples of such libraries preferably include those in which light chainvariable regions constructed as a randomized variable region sequencelibrary or light chain variable regions having germ line sequences arecombined with the sequence of a heavy chain variable region in which aparticular residue(s) has been substituted with at least one amino acidresidue that alters the antigen-binding activity of an antigen-bindingmolecule depending on calcium ion concentrations. Non-limiting examplesof such amino acid residues include amino acid residues of the heavychain CDR1. Further non-limiting examples of such amino acid residuesinclude amino acid residues of the heavy chain CDR2. In addition,non-limiting examples of such amino acid residues also include aminoacid residues of the heavy chain CDR3. Non-limiting examples of suchamino acid residues of heavy chain CDR3 include the amino acids ofpositions 95, 96, 100a, and/or 101 in the CDR3 of heavy chain variableregion as indicated by the Kabat numbering. Furthermore, these aminoacid residues can be contained alone or in combination as long as theyform a calcium-binding motif and/or the antigen-binding activity of anantigen-binding molecule varies depending on calcium ion concentrations.

When light chain variable regions constructed as a randomized variableregion sequence library or light chain variable regions having germ linesequence are combined with a heavy chain variable region into which atleast one amino acid residue that alter the antigen-binding activity ofan antigen-binding molecule depending on ion concentrations as mentionedabove has been introduced, the sequence of the heavy chain variableregion can also be designed to contain flexible residues in the samemanner as described above. The number and position of flexible residuesare not particularly limited as long as the antigen-binding activity ofan antigen-binding molecule of the present invention varies depending onion concentrations. Specifically, the heavy chain CDR and/or FRsequences may contain one or more flexible residues.

Furthermore, randomized variable region libraries can be preferably usedas amino acid sequences of CDR1, CDR2, and/or CDR3 of the heavy chainvariable region other than the amino acid residues that alter theantigen-binding activity of an antigen-binding molecule. When germ linesequences are used as light chain variable regions, non-limitingexamples of such sequences include those of SEQ ID NO: 5 (Vk1), SEQ IDNO: 6 (Vk2), SEQ ID NO: 7 (Vk3), and SEQ ID NO: 8 (Vk4).

Any of the above-described amino acids that alter the antigen-bindingactivity of an antigen-binding molecule depending on calcium ionconcentrations can be preferably used, as long as they form acalcium-binding motif. Specifically, such amino acids includeelectron-donating amino acids. Preferred examples of suchelectron-donating amino acids include, serine, threonine, asparagine,glutamic acid, aspartic acid, and glutamic acid.

Condition of Hydrogen Ion Concentrations

In an embodiment of the present invention, the condition of ionconcentrations refers to the condition of hydrogen ion concentrations orpH condition. In the present invention, the concentration of proton,i.e., the nucleus of hydrogen atom, is treated as synonymous withhydrogen index (pH). When the activity of hydrogen ion in an aqueoussolution is represented as aH+, pH is defined as −log 10aH+. When theionic strength of the aqueous solution is low (for example, lower than10⁻³), aH+ is nearly equal to the hydrogen ion strength. For example,the ionic product of water at 25° C. and 1 atmosphere isKw=aH+aOH=10⁻¹⁴, and therefore in pure water, aH+=aOH=10⁻⁷. In thiscase, pH=7 is neutral; an aqueous solution whose pH is lower than 7 isacidic or whose pH is greater than 7 is alkaline.

In the present invention, when pH condition is used as the ionconcentration condition, pH conditions include high hydrogen ionconcentrations or low pHs, i.e., an acidic pH range, and low hydrogenion concentrations or high pHs, i.e., a neutral pH range. “The bindingactivity varies depending on pH condition” means that theantigen-binding activity of an antigen-binding molecule varies due tothe difference in conditions of a high hydrogen ion concentration or lowpH (an acidic pH range) and a low hydrogen ion concentration or high pH(a neutral pH range). This includes, for example, the case where theantigen-binding activity of an antigen-binding molecule is higher in aneutral pH range than in an acidic pH range and the case where theantigen-binding activity of an antigen-binding molecule is higher in anacidic pH range than in a neutral pH range.

Herein, neutral pH range is not limited to a specific value and ispreferably selected from between pH 6.7 and pH 10.0. In anotherembodiment, the pH can be selected from between pH 6.7 and pH 9.5. Instill another embodiment, the pH can be selected from between pH 7.0 andpH 9.0. In yet another embodiment, the pH can be selected from betweenpH 7.0 and pH 8.0. In particular, the preferred pH includes pH 7.4,which is close to the pH of plasma (blood) in vivo.

Herein, an acidic pH range is not limited to a specific value and ispreferably selected from between pH 4.0 and pH 6.5. In anotherembodiment, the pH can be selected from between pH 4.5 and pH 6.5. Instill another embodiment, the pH can be selected from between pH 5.0 andpH 6.5. In yet another embodiment, the pH can be selected from betweenpH 5.5 and pH 6.5. In particular, the preferred pH includes pH 5.8,which is close to the ionized calcium concentration in the earlyendosome in vivo.

In the present invention, “the antigen-binding activity of anantigen-binding molecule at a high hydrogen ion concentration or low pH(an acidic pH range) is lower than that at a low hydrogen ionconcentration or high pH (a neutral pH range)” means that theantigen-binding activity of an antigen-binding molecule at a pH selectedfrom between pH 4.0 and pH 6.5 is weaker than that at a pH selected frombetween pH 6.7 and pH 10.0; preferably means that the antigen-bindingactivity of an antigen-binding molecule at a pH selected from betweenpH4.5 and pH 6.5 is weaker than that at a pH selected from between pH6.7 and pH 9.5; more preferably, means that the antigen-binding activityof an antigen-binding molecule at a pH selected from between pH 5.0 andpH 6.5 is weaker than that at a pH selected from between pH 7.0 and pH9.0; still more preferably means that the antigen-binding activity of anantigen-binding molecule at a pH selected from between pH 5.5 and pH 6.5is weaker than that at a pH selected from between pH 7.0 and pH 8.0;particularly preferably means that the antigen-binding activity at thepH in the early endosome in vivo is weaker than the antigen-bindingactivity at the pH of plasma in vivo; and specifically means that theantigen-binding activity of an antigen-binding molecule at pH 5.8 isweaker than the antigen-binding activity at pH 7.4.

Whether the antigen-binding activity of an antigen-binding molecule haschanged by the pH condition can be determined, for example, by the useof known measurement methods such as those described in the section“Binding Activity” above. Specifically, the binding activity is measuredunder different pH conditions using the measurement methods describedabove. For example, the antigen-binding activity of an antigen-bindingmolecule is compared under the conditions of acidic pH range and neutralpH range to confirm that the antigen-binding activity of theantigen-binding molecule changes to be higher under the condition ofneutral pH range than that under the condition of acidic pH range.

Furthermore, in the present invention, the expression “theantigen-binding activity at a high hydrogen ion concentration or low pH,i.e., in an acidic pH range, is lower than that at a low hydrogen ionconcentration or high pH, i.e., in a neutral pH range” can also beexpressed as “the antigen-binding activity of an antigen-bindingmolecule at a low hydrogen ion concentration or high pH, i.e., in aneutral pH range, is higher than that at a high hydrogen ionconcentration or low pH, i.e., in an acidic pH range”. In the presentinvention, “the antigen-binding activity at a high hydrogen ionconcentration or low pH, i.e., in an acidic pH range, is lower than thatat a low hydrogen ion concentration or high pH, i.e., in a neutral pHrange” may be described as “the antigen-binding activity at a highhydrogen ion concentration or low pH, i.e., in an acidic pH range, isweaker than the antigen-binding ability at a low hydrogen ionconcentration or high pH, i.e., in a neutral pH range”. Alternatively,“the antigen-binding activity at a high hydrogen ion concentration orlow pH, i.e., in an acidic pH range, is reduced to be lower than that ata low hydrogen ion concentration or high pH, i.e., in a neutral pHrange” may be described as “the antigen-binding activity at a highhydrogen ion concentration or low pH, i.e., in an acidic pH range, isreduced to be weaker than the antigen-binding ability at a low hydrogenion concentration or high pH, i.e., in a neutral pH range”.

The conditions other than hydrogen ion concentration or pH for measuringthe antigen-binding activity may be suitably selected by those skilledin the art and are not particularly limited. Measurements can be carriedout, for example, at 37° C. using HEPES buffer. Measurements can becarried out, for example, using Biacore (GE Healthcare). When theantigen is a soluble antigen, the antigen-binding activity of anantigen-binding molecule can be determined by assessing the bindingactivity to the soluble antigen by pouring the antigen as an analyteinto a chip immobilized with the antigen-binding molecule. When theantigen is a membrane antigen, the binding activity to the membraneantigen can be assessed by pouring the antigen-binding molecule as ananalyte into a chip immobilized with the antigen.

As long as the antigen-binding activity of an antigen-binding moleculeof the present invention at a high hydrogen ion concentration or low pH,i.e., in an acidic pH range is weaker than that at a low hydrogen ionconcentration or high pH, i.e., in a neutral pH range, the ratio of theantigen-binding activity between that at a high hydrogen ionconcentration or low pH, i.e., an acidic pH range, and at a low hydrogenion concentration or high pH, i.e., a neutral pH range is notparticularly limited, and the value of KD (pH 5.8)/KD (pH 7.4), which isthe ratio of the dissociation constant (KD) for an antigen at a highhydrogen ion concentration or low pH, i.e., in an acidic pH range to theKD at a low hydrogen ion concentration or high pH, i.e., in a neutral pHrange, is preferably 2 or more; more preferably the value of KD (pH5.8)/KD (pH 7.4) is 10 or more; and still more preferably the value ofKD (pH 5.8)/KD (pH 7.4) is 40 or more. The upper limit of KD (pH 5.8)/KD(pH 7.4) value is not particularly limited, and may be any value such as400, 1000, or 10000, as long as the molecule can be produced by thetechniques of those skilled in the art.

When the antigen is a soluble antigen, the dissociation constant (KD)can be used as the value for antigen-binding activity. Meanwhile, whenthe antigen is a membrane antigen, the apparent dissociation constant(KD) can be used. The dissociation constant (KD) and apparentdissociation constant (KD) can be measured by methods known to thoseskilled in the art, and Biacore (GE healthcare), Scatchard plot, flowcytometer, and such can be used.

Alternatively, for example, the dissociation rate constant (kd) can besuitably used as an index for indicating the ratio of theantigen-binding activity of an antigen-binding molecule of the presentinvention between that at a high hydrogen ion concentration or low pH,i.e., an acidic pH range and a low hydrogen ion concentration or highpH, i.e., a neutral pH range. When kd (dissociation rate constant) isused as an index for indicating the binding activity ratio instead of KD(dissociation constant), the value of kd (in an acidic pH range)/kd (ina neutral pH range), which is the ratio of kd (dissociation rateconstant) for the antigen at a high hydrogen ion concentration or lowpH, i.e., in an acidic pH range to kd (dissociation rate constant) at alow hydrogen ion concentration or high pH, i.e., in a neutral pH range,is preferably 2 or more, more preferably 5 or more, still morepreferably 10 or more, and yet more preferably 30 or more. The upperlimit of kd (in an acidic pH range)/kd (in a neutral pH range) value isnot particularly limited, and may be any value such as 50, 100, or 200,as long as the molecule can be produced by the techniques of thoseskilled in the art.

When the antigen is a soluble antigen, the dissociation rate constant(kd) can be used as the value for antigen-binding activity and when theantigen is a membrane antigen, the apparent dissociation rate constant(kd) can be used. The dissociation rate constant (kd) and apparentdissociation rate constant (kd) can be determined by methods known tothose skilled in the art, and Biacore (GE healthcare), flow cytometer,and such may be used. In the present invention, when the antigen-bindingactivity of an antigen-binding molecule is measured at differenthydrogen ion concentrations, i.e., pHs, conditions other than thehydrogen ion concentration, i.e., pH, are preferably the same.

For example, an antigen-binding domain or antibody whose antigen-bindingactivity at a high hydrogen ion concentration or low pH, i.e., in anacidic pH range is lower than that at a low hydrogen ion concentrationor high pH, i.e., in a neutral pH range, which is one embodimentprovided by the present invention, can be obtained via screening ofantigen-binding domains or antibodies, comprising the following steps(a) to (c):

(a) obtaining the antigen-binding activity of an antigen-binding domainor antibody in an acidic pH range;(b) obtaining the antigen-binding activity of an antigen-binding domainor antibody in a neutral pH range; and(c) selecting an antigen-binding domain or antibody whoseantigen-binding activity in the acidic pH range is lower than that inthe neutral pH range.

Alternatively, an antigen-binding domain or antibody whoseantigen-binding activity at a high hydrogen ion concentration or low pH,i.e., in an acidic pH range, is lower than that at a low hydrogen ionconcentration or high pH, i.e., in a neutral pH range, which is oneembodiment provided by the present invention, can be obtained viascreening of antigen-binding domains or antibodies, or a librarythereof, comprising the following steps (a) to (c):

(a) contacting an antigen-binding domain or antibody, or a librarythereof, in a neutral pH range with an antigen;(b) placing in an acidic pH range the antigen-binding domain or antibodybound to the antigen in step (a); and(c) isolating the antigen-binding domain or antibody dissociated in step(b).

An antigen-binding domain or antibody whose antigen-binding activity ata high hydrogen ion concentration or low pH, i.e., in an acidic pH rangeis lower than that at a low hydrogen ion concentration or high pH, i.e.,in a neutral pH range, which is another embodiment provided by thepresent invention, can be obtained via screening of antigen-bindingdomains or antibodies, or a library thereof, comprising the followingsteps (a) to (d):

(a) contacting in an acidic pH range an antigen with a library ofantigen-binding domains or antibodies;(b) selecting the antigen-binding domain or antibody which does not bindto the antigen in step (a);(c) allowing the antigen-binding domain or antibody selected in step (b)to bind with the antigen in a neutral pH range; and(d) isolating the antigen-binding domain or antibody bound to theantigen in step (c).

An antigen-binding domain or antibody whose antigen-binding activity ata high hydrogen ion concentration or low pH, i.e., in an acidic pHrange, is lower than that at a low hydrogen ion concentration or highpH, i.e., in a neutral pH range, which is even another embodimentprovided by the present invention, can be obtained by a screening methodcomprising the following steps (a) to (c):

(a) contacting in a neutral pH range a library of antigen-bindingdomains or antibodies with a column immobilized with an antigen;(b) eluting in an acidic pH range from the column the antigen-bindingdomain or antibody bound to the column in step (a); and(c) isolating the antigen-binding domain or antibody eluted in step (b).

An antigen-binding domain or antibody whose antigen-binding activity ata high hydrogen ion concentration or low pH, i.e., in an acidic pH,range is lower than that at a low hydrogen ion concentration or high pH,i.e., in a neutral pH range, which is still another embodiment providedby the present invention, can be obtained by a screening methodcomprising the following steps (a) to (d):

(a) allowing, in an acidic pH range, a library of antigen-bindingdomains or antibodies to pass a column immobilized with an antigen;(b) collecting the antigen-binding domain or antibody eluted withoutbinding to the column in step (a);(c) allowing the antigen-binding domain or antibody collected in step(b) to bind with the antigen in a neutral pH range; and(d) isolating the antigen-binding domain or antibody bound to theantigen in step (c).

An antigen-binding domain or antibody whose antigen-binding activity ata high hydrogen ion concentration or low pH, i.e., in an acidic pHrange, is lower than that at a low hydrogen ion concentration or highpH, i.e., in a neutral pH range, which is yet another embodimentprovided by the present invention, can be obtained by a screening methodcomprising the following steps (a) to (d):

(a) contacting an antigen with a library of antigen-binding domains orantibodies in a neutral pH range;(b) obtaining the antigen-binding domain or antibody bound to theantigen in step (a);(c) placing in an acidic pH range the antigen-binding domain or antibodyobtained in step (b); and(d) isolating the antigen-binding domain or antibody whoseantigen-binding activity in step (c) is weaker than the criterion forthe selection in step (b).

The above-described steps may be repeated twice or more times. Thus, thepresent invention provides antigen-binding domains and antibodies whoseantigen-binding activity in an acidic pH range is lower than that in aneutral pH range, which are obtained by a screening method that furthercomprises the steps of repeating steps (a) to (c) or (a) to (d) in theabove-described screening methods. The number of times that steps (a) to(c) or (a) to (d) is repeated is not particularly limited; however, thenumber is 10 or less in general.

In the screening methods of the present invention, the antigen-bindingactivity of an antigen-binding domain or antibody at a high hydrogen ionconcentration or low pH, i.e., in an acidic pH range, is notparticularly limited, as long as it is the antigen-binding activity at apH of between 4.0 and 6.5, and includes the antigen-binding activity ata pH of between 4.5 and 6.6 as the preferred pH. The antigen-bindingactivity also includes that at a pH of between 5.0 and 6.5, and that ata pH of between 5.5 and 6.5 as another preferred pH. The antigen-bindingactivity also includes that at the pH in the early endosome in vivo asthe more preferred pH, and specifically, that at pH 5.8. Meanwhile, theantigen-binding activity of an antigen-binding domain or antibody at alow hydrogen ion concentration or high pH, i.e., in a neutral pH range,is not particularly limited, as long as it is the antigen-bindingactivity at a pH of between 6.7 and 10, and includes the antigen-bindingactivity at a pH of between 6.7 and 9.5 as the preferred pH. Theantigen-binding activity also includes that at a pH of between 7.0 and9.5 and that at a pH of between 7.0 and 8.0 as another preferred pH. Theantigen-binding activity also includes that at the pH of plasma in vivoas the more preferred pH, and specifically, that at pH 7.4.

The antigen-binding activity of an antigen-binding domain or antibodycan be measured by methods known to those skilled in the art. Thoseskilled in the art can suitably determine conditions other than ionizedcalcium concentration. The antigen-binding activity of anantigen-binding domain or antibody can be assessed based on thedissociation constant (KD), apparent dissociation constant (KD),dissociation rate constant (kd), apparent dissociation rate constant(kd), and such. These can be determined by methods known to thoseskilled in the art, for example, using Biacore (GE healthcare),Scatchard plot, or FACS.

Herein, the step of selecting an antigen-binding domain or antibodywhose antigen-binding activity at a low hydrogen ion concentration orhigh pH, i.e., in a neutral pH range, is higher than that at a highhydrogen ion concentration or low pH, i.e., in an acidic pH range, issynonymous with the step of selecting an antigen-binding domain orantibody whose antigen-binding activity at a high hydrogen ionconcentration or low pH, i.e., in an acidic pH range, is lower than thatat a low hydrogen ion concentration or high pH, i.e., in a neutral pHrange.

As long as the antigen-binding activity at a low hydrogen ionconcentration or high pH, i.e., in a neutral pH range, is higher thanthat at a high hydrogen ion concentration or low pH, i.e., in an acidicpH range, the difference between the antigen-binding activity at a lowhydrogen ion concentration or high pH, i.e., a neutral pH range, andthat at a high hydrogen ion concentration or low pH, i.e., an acidic pHrange, is not particularly limited; however, the antigen-bindingactivity at a low hydrogen ion concentration or high pH, i.e., in aneutral pH range, is preferably twice or more, more preferably 10 timesor more, and still more preferably 40 times or more than that at a highhydrogen ion concentration or low pH, i.e., in an acidic pH range.

The antigen binding domain or antibody of the present invention screenedby the screening methods described above may be any antigen-bindingdomain or antibody, and the above-mentioned antigen-binding domain orantibody may be screened. For example, antigen-binding domain orantibody having the native sequence may be screened, and antigen-bindingdomain or antibody in which their amino acid sequences have beensubstituted may be screened.

The antigen-binding domain or antibody of the present invention to bescreened by the above-described screening methods may be prepared in anymanner. For example, conventional antibodies, conventional libraries(phage library, etc.), antibodies or libraries prepared from B cells ofimmunized animals or from hybridomas obtained by immunizing animals,antibodies or libraries (libraries with increased content of amino acidswith a side chain pKa of 4.0-8.0 (for example, histidine and glutamicacid) or unnatural amino acids, libraries introduced with amino acidswith a side chain pKa of 4.0-8.0 (for example, histidine and glutamicacid) or unnatural amino acid mutations at specific positions, etc.)obtained by introducing amino acids with a side chain pKa of 4.0-8.0(for example, histidine and glutamic acid) or unnatural amino acidmutations into the above-described antibodies or libraries may be used.

Methods for obtaining an antigen-binding domain or antibody whoseantigen-binding activity at a low hydrogen ion concentration or high pH,i.e., in a neutral pH range, is higher than that at a high hydrogen ionconcentration or low pH, i.e., in an acidic pH range, from anantigen-binding domains or antibodies prepared from hybridomas obtainedby immunizing animals or from B cells of immunized animals preferablyinclude, for example, the antigen-binding molecule or antibody in whichat least one of the amino acids of the antigen-binding domain orantibody is substituted with an amino acid with a side chain pKa of4.0-8.0 (for example, histidine and glutamic acid) or an unnatural aminoacid mutation, or the antigen-binding domain or antibody inserted withan amino acid with a side chain pKa of 4.0-8.0 (for example, histidineand glutamic acid) or unnatural amino acid, such as those described inWO 2009/125825.

The sites of introducing mutations of amino acids with a side chain pKaof 4.0-8.0 (for example, histidine and glutamic acid) or unnatural aminoacids are not particularly limited, and may be any position as long asthe antigen-binding activity in an acidic pH range becomes weaker thanthat in a neutral pH range (the value of KD (in an acidic pH range)/KD(in a neutral pH range) or kd (in an acidic pH range)/kd (in a neutralpH range) is increased) as compared to before substitution or insertion.For example, when the antigen-binding molecule is an antibody, antibodyvariable region and CDRs are suitable. Those skilled in the art canappropriately determine the number of amino acids to be substituted withor the number of amino acids with a side chain pKa of 4.0-8.0 (forexample, histidine and glutamic acid) or unnatural amino acids to beinserted. It is possible to substitute with a single amino acid having aside chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) ora single unnatural amino acid; it is possible to insert a single aminoacid having a side chain pKa of 4.0-8.0 (for example, histidine andglutamic acid) or a single unnatural amino acid; it is possible tosubstitute with two or more amino acids having a side chain pKa of4.0-8.0 (for example, histidine and glutamic acid) or two or moreunnatural amino acids; and it is possible to insert two or more aminoacids having a side chain pKa of 4.0-8.0 (for example, histidine andglutamic acid) or two or more unnatural amino acids. Alternatively,other amino acids can be deleted, added, inserted, and/or substitutedconcomitantly, aside from the substitution into amino acids having aside chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) orunnatural amino acids, or the insertion of amino acids having a sidechain pKa of 4.0-8.0 (for example, histidine and glutamic acid) orunnatural amino acids. Substitution into or insertion of amino acidswith a side chain pKa of 4.0-8.0 (for example, histidine and glutamicacid) or unnatural amino acids can performed randomly by methods such ashistidine scanning, in which the alanine of alanine scanning known tothose skilled in the art is replaced with histidine. Antigen-bindingmolecules exhibiting a greater value of KD (in an acidic pH range)/KD(in a neutral pH range) or kd (in an acidic pH range)/kd (in a neutralpH range) as compared to before the mutation can be selected fromantigen-binding domains or antibodies introduced with random insertionsor substitution mutations of amino acids with a side chain pKa of4.0-8.0 (for example, histidine and glutamic acid) or unnatural aminoacids.

Preferred examples of antigen-binding molecules containing the mutationinto amino acids with a side chain pKa of 4.0-8.0 (for example,histidine and glutamic acid) or unnatural amino acids as described aboveand whose antigen-binding activity in an acidic pH range is lower thanthat in a neutral pH range include, antigen-binding molecules whoseantigen-binding activity in the neutral pH range after the mutation intoamino acids with a side chain pKa of 4.0-8.0 (for example, histidine andglutamic acid) or unnatural amino acids is comparable to that before themutation into amino acids with a side chain pKa of 4.0-8.0 (for example,histidine and glutamic acid) or unnatural amino acids. Herein, “anantigen-binding molecule after the mutation with amino acids having aside chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) orunnatural amino acids has an antigen-binding activity comparable to thatbefore the mutation with amino acids having a side chain pKa of 4.0-8.0(for example, histidine and glutamic acid) or unnatural amino acids”means that, when taking the antigen-binding activity of anantigen-binding molecule before the mutation with amino acids having aside chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) orunnatural amino acids as 100%, the antigen-binding activity of anantigen-binding molecule after the mutation with amino acids having aside chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) orunnatural amino acids is at least 10% or more, preferably 50% or more,more preferably 80% or more, and still more preferably 90% or more. Theantigen-binding activity after the mutation of amino acids with a sidechain pKa of 4.0-8.0 (for example, histidine and glutamic acid) orunnatural amino acids at pH 7.4 may be higher than that before themutation of amino acids with a side chain pKa of 4.0-8.0 (for example,histidine and glutamic acid) or unnatural amino acids at pH 7.4. If theantigen-binding activity of an antigen-binding molecule is decreased dueto insertion of or substitution into amino acids with a side chain pKaof 4.0-8.0 (for example, histidine and glutamic acid) or unnatural aminoacids, the antigen-binding activity can be made to be comparable to thatbefore the insertion of or substitution into amino acids with a sidechain pKa of 4.0-8.0 (for example, histidine and glutamic acid) orunnatural amino acids, by introducing a substitution, deletion,addition, and/or insertion of one or more amino acids of theantigen-binding molecule. The present invention also includesantigen-binding molecules whose binding activity has been adjusted to becomparable by substitution, deletion, addition, and/or insertion of oneor more amino acids after substitution or insertion of amino acids witha side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid)or unnatural amino acids. Meanwhile, when an antigen-binding molecule isa substance containing an antibody constant region, preferredembodiments of antigen-binding molecules whose antigen-binding activityat an acidic pH range is lower than that in a neutral pH range includemethods in which the antibody constant regions contained in theantigen-binding molecules have been modified. Specific examples ofmodified antibody constant regions preferably include the constantregions of SEQ ID NOs: 11, 12, 13, and 14.

Amino Acids that Alter the Antigen-Binding Activity of Antigen-BindingDomain Depending on the Hydrogen Ion Concentration Conditions

Antigen-binding domains or antibodies of the present invention to bescreened by the above-described screening methods may be prepared in anymanner. For example, when ion concentration condition is hydrogen ionconcentration condition or pH condition, conventional antibodies,conventional libraries (phage library, etc.), antibodies or librariesprepared from B cells of immunized animals or from hybridomas obtainedby immunizing animals, antibodies or libraries (libraries with increasedcontent of amino acids with a side chain pKa of 4.0-8.0 (for example,histidine and glutamic acid) or unnatural amino acids, librariesintroduced with mutations of amino acids with a side chain pKa of4.0-8.0 (for example, histidine and glutamic acid) or unnatural aminoacids at specific positions, etc.) obtained by introducing mutations ofamino acids with a side chain pKa of 4.0-8.0 (for example, histidine andglutamic acid) or unnatural amino acids into the above-describedantibodies or libraries may be used.

In one non-limiting embodiment of the present invention, a librarycontaining multiple antigen-binding molecules of the present inventionwhose sequences are different from one another can also be constructedby combining heavy chain variable regions, produced as a randomizedvariable region sequence library, with light chain variable regionsintroduced with “at least one amino acid residue that changes theantigen-binding activity of an antigen-binding molecule depending on thehydrogen ion concentration condition”.

Such amino acid residues include, but are not limited to, for example,amino acid residues contained in the light chain CDR1. The amino acidresidues also include, but are not limited to, for example, amino acidresidues contained in the light chain CDR2. The amino acid residues alsoinclude, but are not limited to, for example, amino acid residuescontained in the light chain CDR3.

The above-described amino acid residues contained in the light chainCDR1 include, but are not limited to, for example, amino acid residuesof positions 24, 27, 28, 31, 32, and/or 34 according to Kabat numberingin the CDR1 of light chain variable region. Meanwhile, the amino acidresidues contained in the light chain CDR2 include, but are not limitedto, for example, amino acid residues of positions 50, 51, 52, 53, 54,55, and/or 56 according to Kabat numbering in the CDR2 of light chainvariable region. Furthermore, the amino acid residues in the light chainCDR3 include, but are not limited to, for example, amino acid residuesof positions 89, 90, 91, 92, 93, 94, and/or 95A according to Kabatnumbering in the CDR3 of light chain variable region. Moreover, theamino acid residues can be contained alone or can be contained incombination of two or more amino acids as long as they allow the changein the antigen-binding activity of an antigen-binding molecule dependingon the hydrogen ion concentration.

Even when the heavy chain variable region produced as a randomizedvariable region sequence library is combined with the above-describedlight chain variable region introduced with “at least one amino acidresidue that changes the antigen-binding activity of an antigen-bindingmolecule depending on the hydrogen ion concentration condition”, it ispossible to design so that the flexible residues are contained in thesequence of the light chain variable region in the same manner asdescribed above. The number and position of the flexible residues arenot particularly limited to a specific embodiment, as long as theantigen-binding activity of an antigen-binding molecule of the presentinvention changes depending on the hydrogen ion concentration condition.Specifically, the CDR and/or FR sequences of heavy chain and/or lightchain can contain one or more flexible residues. For example, flexibleresidues to be introduced into the sequences of the light chain variableregions include, but are not limited to, for example, the amino acidresidues listed in Tables 3 and 4. Meanwhile, amino acid sequences oflight chain variable regions other than the flexible residues and aminoacid residues that change the antigen-binding activity of anantigen-binding molecule depending on the hydrogen ion concentrationcondition suitably include, but are not limited to, germ line sequencessuch as Vk1 (SEQ ID NO: 5), Vk2 (SEQ ID NO: 6), Vk3 (SEQ ID NO: 7), andVk4 (SEQ ID NO: 8).

TABLE 3 POSITION AMINO ACID CDR1 28 S: 100% 29 I: 100% 30 N: 25% S: 25%R: 25% H: 25% 31 S: 100% 32 H: 100% 33 L: 100% 34 A: 50% N: 50% CDR2 50H: 100% OR A: 25% D: 25% G: 25% K: 25% 51 A: 100% A: 100% 52 S: 100% S:100% 53 K: 33.3% N: 33.3% S: 33.3% H: 100% 54 L: 100% L: 100% 55 Q: 100%Q: 100% 56 S: 100% S: 100% CDR3 90 Q: 100% OR Q: 100% 91 H: 100% S:33.3% R: 33.3% Y: 33.3% 92 G: 25% N: 25% S: 25% Y: 25% H: 100% 93 H:33.3% N: 33.3% S: 33.3% H: 33.3% N: 33.3% S: 33.3% 94 S: 50% Y: 50% S:50% Y: 50% 95 P: 100% P: 100% 96 L: 50% Y: 50% L: 50% Y: 50% (Positionindicates Kabat numbering)

TABLE 4 CDR POSITION AMINO ACID CDR1 28 S: 100% 29 I: 100% 30 H: 30% N:10% S: 50% R: 10% 31 N: 35% S: 65% 32 H: 40% N: 20% Y: 40% 33 L: 100% 34A: 70% N: 30% CDR2 50 A: 25% D: 15% G: 25% H: 30% K: 5% 51 A: 100% 52 S:100% 53 H: 30% K: 10% N: 15% S: 45% 54 L: 100% 55 Q: 100% 56 S: 100%CDR3 90 Q: 100% 91 H: 30% S: 15% R: 10% Y: 45% 92 G: 20% H: 30% N: 20%S: 15% Y: 15% 93 H: 30% N: 25% S: 45% 94 S: 50% Y: 50% 95 P: 100% 96 L:50% Y: 50% (Position indicates Kabat numbering)

Any amino acid residue may be suitably used as the above-described aminoacid residues that change the antigen-binding activity of anantigen-binding molecule depending on the hydrogen ion concentrationcondition. Specifically, such amino acid residues include amino acidswith a side chain pKa of 4.0-8.0. Such electron-releasing amino acidspreferably include, for example, naturally occurring amino acids such ashistidine and glutamic acid, as well as unnatural amino acids such ashistidine analogs (US20090035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr(pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11(17), 3761-2768). Particularly preferred amino acid residues include,for example, amino acids with a side chain pKa of 6.0-7.0. Suchelectron-releasing amino acid residues preferably include, for example,histidine.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc.Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap extension PCR canbe appropriately employed to modify the amino acids of antigen-bindingdomains. Furthermore, various known methods can also be used as an aminoacid modification method for substituting amino acids by those otherthan natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35,225-249; Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). Forexample, a cell-free translation system (Clover Direct (ProteinExpress)) containing tRNAs in which amber suppressor tRNA, which iscomplementary to UAG codon (amber codon) that is a stop codon, is linkedwith an unnatural amino acid may be suitably used.

The preferred heavy chain variable region that is used in combinationincludes, for example, randomized variable region libraries. Knownmethods are appropriately combined as a method for producing arandomized variable region library. In a non-limiting embodiment of thepresent invention, an immune library constructed based on antibody genesderived from animals immunized with specific antigens, patients withinfection or persons with an elevated antibody titer in blood as aresult of vaccination, cancer patients, or lymphocytes of auto immunediseases may be suitably used as a randomized variable region library.

In another non-limiting embodiment of the present invention, in the samemanner as described above, a synthetic library in which the CDRsequences of V genes from genomic DNA or functional reconstructed Vgenes are replaced with a set of synthetic oligonucleotides containingthe sequences encoding codon sets of an appropriate length can also besuitably used as a randomized variable region library. In this case, theCDR3 sequence alone may be replaced because variety in the gene sequenceof heavy chain CDR3 is observed. The basis for giving rise to amino acidvariations in the variable region of an antigen-binding molecule is togenerate variations of amino acid residues of surface-exposed positionsof the antigen-binding molecule. The surface-exposed position refers toa position where an amino acid is exposed on the surface and/orcontacted with an antigen based on the conformation, structuralensemble, and/or modeled structure of an antigen-binding molecule, andin general, such positions are the CDRs. The surface-exposed positionsare preferably determined using the coordinates derived from athree-dimensional model of the antigen-binding molecule using computerprograms such as InsightII program (Accelrys). The surface-exposedpositions can be determined using algorithms known in the art (forexample, Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly(J. Appl. Cryst. (1983) 16, 548-558)). The surface-exposed positions canbe determined based on the information on the three dimensionalstructure of antibodies using software suitable for protein modeling.Software which is suitably used for this purpose includes the SYBYLbiopolymer module software (Tripos Associates). When the algorithmrequires the input size parameter from the user, the “size” of probe foruse in computation is generally or preferably set at about 1.4 angstromor less in radius. Furthermore, a method for determining surface-exposedregion and area using PC software is described by Pacios (Comput. Chem.(1994) 18 (4), 377-386; and J. Mol. Model. (1995) 1, 46-53).

In still another non-limiting embodiment of the present invention, anaive library constructed from antibody genes derived from lymphocytesof healthy persons and consisting of naive sequences, which are unbiasedrepertoire of antibody sequences, can also be particularly suitably usedas a randomized variable region library (Gejima et al. (Human Antibodies(2002) 11, 121-129); and Cardoso et al. (Scand. J. Immunol. (2000) 51,337-344)).

FcRn

Unlike Fcγ receptor belonging to the immunoglobulin superfamily, FcRn,human FcRn in particular, is structurally similar to polypeptides ofmajor histocompatibility complex (MHC) class I, exhibiting 22% to 29%sequence identity to class I MHC molecules (Ghetie el al., Immunol.Today (1997) 18 (12): 592-598). FcRn is expressed as a heterodimerconsisting of soluble β or light chain (β2 microglobulin) complexed withtransmembrane a or heavy chain. Like MHC, FcRn α chain comprises threeextracellular domains (α1, α2, and α3) and its short cytoplasmic domainanchors the protein onto the cell surface. α1 and α2 domains interactwith the FcRn-binding domain of the antibody Fc region (Raghavan et al.,Immunity (1994) 1: 303-315).

FcRn is expressed in maternal placenta and york sac of mammals, and isinvolved in mother-to-fetus IgG transfer. In addition, in neonatal smallintestine of rodents, where FcRn is expressed, FcRn is involved intransfer of maternal IgG across brush border epithelium from ingestedcolostrum or milk. FcRn is expressed in a variety of other tissues andendothelial cell systems of various species. FcRn is also expressed inadult human endothelia, muscular blood vessels, and hepatic sinusoidalcapillaries. FcRn is believed to play a role in maintaining the plasmaIgG concentration by mediating recycling of IgG to serum upon binding toIgG. Typically, binding of FcRn to IgG molecules is strictly pHdependent. The optimal binding is observed in an acidic pH range below7.0.

Human FcRn whose precursor is a polypeptide having the signal sequenceof SEQ ID NO: 15 (the polypeptide with the signal sequence is shown inSEQ ID NO: 16) forms a complex with human β2-microglobulin in vivo. Asshown in the Reference Examples described below, soluble human FcRncomplexed with β2-microglobulin is produced by using conventionalrecombinant expression techniques. FcRn regions of the present inventioncan be assessed for their binding activity to such a soluble human FcRncomplexed with β2-microglobulin. Herein, unless otherwise specified,human FcRn refers to a form capable of binding to an FcRn region of thepresent invention. Examples include a complex between human FcRn andhuman β2-microglobulin.

FcRn-Binding Domains

An embodiment of the present invention provides pharmaceuticalcompositions that induce an immune response to the aforementionedantigen, which comprises as an active ingredient an antigen-bindingmolecule containing an antigen-binding domain whose antigen-bindingactivity changes depending on ion concentration conditions and anFcRn-binding domain having FcRn-binding activity in a neutral pH range.

Antigen-binding molecules of the present invention have an FcRn-bindingdomain. The FcRn-binding domain is not particularly limited as long asthe antigen-binding molecule has FcRn-binding activity in a neutral pHrange, and it may be a domain that has activity of directly orindirectly binding to FcRn. Preferred examples of such domains includeFc regions of IgG immunoglobulins, albumin, albumin domain 3, anti-FcRnantibodies, anti-FcRn peptides, anti-FcRn scaffold molecules, and such,which have activity of directly binding to FcRn, or molecules that bindto IgG or albumin, which have activity of indirectly binding to FcRn.For the anti-FcRn scaffold, it is possible to use a domain with anystructure of the aforementioned antigen-binding domains characterized bybinding to FcRn. In the present invention, a domain having bindingactivity to FcRn in an acidic pH range and neutral pH range arepreferred. Such a domain may be preferably used as it is if it is adomain already having FcRn-binding activity in a neutral pH range. Whenthe domain has no or weak FcRn-binding activity in a neutral pH range,amino acids in the antigen-binding molecule can be modified to impartFcRn-binding activity. Alternatively, FcRn binding activity may beenhanced by altering amino acids in the domain already havingFcRn-binding activity in a neutral pH range. Desired amino acidalterations in the FcRn-binding domain can be identified by comparingthe FcRn-binding activity in a neutral pH range before and after aminoacid alteration.

Furthermore, in a different embodiment of the present invention, an FcRnbinding domain that has FcRn-binding activity under a low calcium ionconcentration condition and high calcium ion concentration condition ispreferably used. Such a domain may be preferably used as it is if thedomain already has FcRn-binding activity under a high calcium ionconcentration condition. When the domain has no or weak FcRn-bindingactivity under a high calcium ion concentration condition, amino acidsin the antigen-binding molecule can be modified to impart FcRn-bindingactivity. Alternatively, FcRn binding activity may be increased byaltering amino acids in the domain already having FcRn-binding activityunder a high calcium ion concentration condition. Desired amino acidalterations in the FcRn-binding domain can be identified by comparingthe FcRn-binding activity under a high calcium ion concentrationcondition before and after amino acid alteration. FcRn-binding domainsmay be obtained by methods based on the methods of screening orproducing antigen-binding domains having antigen-binding activity thatchanges depending on calcium ion concentration conditions as mentionedabove in the section of “Conditions of ion concentration”. Examples ofsuch FcRn-binding domains include anti-FcRn antibodies, anti-FcRnpeptides, anti-FcRn scaffold molecules, and such.

The preferred human FcRn-binding domain is a region that directly bindsto FcRn. Such preferred FcRn-binding domains include, for example,antibody Fc regions. Meanwhile, regions capable of binding to apolypeptide such as albumin or IgG; which has FcRn-binding activity, canindirectly bind to FcRn via albumin, IgG; or such. Therefore, for theFcRn-binding region in the present invention, a region that binds to apolypeptide having FcRn-binding activity may be preferably used. An Fcregion contains an amino acid sequence derived from the constant regionof an antibody heavy chain. An Fc region is a portion of the antibodyheavy chain constant region beginning from the N terminus of the hingeregion at the papain cleavage site, which is on the amino acid atapproximately position 216 according to EU numbering, and including thehinge, CH2 and CH3 domains.

The binding activity of an FcRn binding domain of the present inventionto FcRn, human FcRn in particular, can be measured by methods known tothose skilled in the art, as described in the section “Binding Activity”above. Those skilled in the art can appropriately determine theconditions other than pH. The antigen-binding activity and humanFcRn-binding activity of an antigen-binding molecule can be assessedbased on the dissociation constant (KD), apparent dissociation constant(KD), dissociation rate (kd), apparent dissociation rate (kd), and such.These can be measured by methods known to those skilled in the art. Forexample, Biacore (GE healthcare), Scatchard plot, or flow cytometer maybe used.

When the human FcRn-binding activity of an FcRn-binding domain ismeasured, conditions other than the pH are not particularly limited, andcan be appropriately selected by those skilled in the art. Measurementscan be carried out, for example, at 37° C. using MES buffer, asdescribed in WO 2009/125825. Alternatively, the FcRn-binding activity ofan FcRn-binding domain can be measured by methods known to those skilledin the art, and may be measured by using, for example, Biacore (GEHealthcare) or such. The binding activity of an FcRn-binding domain toFcRn can be assessed by pouring, as an analyte, FcRn, an FcRn-bindingdomain, or an antigen-binding molecule of the present inventioncontaining the FcRn-binding domain into a chip immobilized with anFcRn-binding domain, an antigen-binding molecule of the presentinvention containing the FcRn-binding domain, or FcRn.

The acidic pH range as a condition under which the FcRn-binding domainin the antigen-binding molecule of the present invention hasFcRn-binding activity usually refers to pH4.0 to pH6.5. It preferablyrefers to pH5.5 to pH6.5, and particularly preferably pH5.8 to pH6.0,which is close to the pH in the early-stage endosome in vivo.Furthermore, the neutral pH range as a condition under which theFcRn-binding domain in the antigen-binding molecule of the presentinvention has FcRn-binding activity usually refers to pH6.7 to pH10.0.The neutral pH range is preferably a range indicated by any pH valuewithin pH7.0 to pH8.0, and is preferably selected from pH7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and is particularlypreferably pH7.4, which is close to the plasma (blood) pH in vivo. Whenthe binding affinity between a human FcRn-binding domain and human FcRnis difficult to evaluate because the binding affinity at pH7.4 is low,pH7.0 can be used instead of pH7.4. For the temperature to be used forthe measurement conditions, the binding affinity between the FcRnbinding domain and FcRn can be evaluated at any temperature from 10° C.to 50° C. Preferably, a temperature from 15° C. to 40° C. is used todetermine the binding affinity between an FcRn-binding domain and humanFcRn. More preferably, any temperature from 20° C. to 35° C., such asany temperature selected from 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, and 35° C., is used similarly to determine thebinding affinity between an FcRn binding domain and FcRn. In anembodiment of the present invention, 25° C. is a non-limiting example ofsuch temperature.

According to the Journal of Immunology (2009) 182: 7663-7671, the humanFcRn-binding activity of naturally occurring human IgG1 is a KD of 1.7μM in the acidic pH range (pH6.0), but hardly detectable in the neutralpH range. Therefore, in a preferred embodiment, antigen-bindingmolecules of the present invention having human FcRn-binding activity inan acidic pH range may be used, which include antigen-binding moleculeswhose human FcRn-binding activity in an acidic pH range is a KD of 20 μMor stronger, and whose human FcRn-binding activity in a neutral pH rangeis equivalent to that of naturally-occurring human IgG In a morepreferred embodiment, antigen-binding molecules of the present inventionincluding antigen-binding molecules whose human FcRn-binding activity inan acidic pH range is a KD of 2.0 μM or stronger may be used. In an evenmore preferred embodiment, antigen-binding molecules whose humanFcRn-binding activity in an acidic pH range is a KD of 0.5 μM orstronger may be used. The above-mentioned KD values are determined bythe method described in the Journal of Immunology (2009) 182: 7663-7671(antigen-binding molecules are immobilized onto a chip and human FcRn isallowed to flow as an analyte).

In the present invention, an Fc region having FcRn-binding activity inan acidic pH range is preferred. If such a domain is an Fc regionalready having FcRn-binding activity in an acidic pH range, it may beused as it is. If the domain has no or weak FcRn-binding activity in anacidic pH range, an Fc region having desired FcRn-binding activity maybe obtained by altering amino acids in the antigen-binding molecule.Also, an Fc region having desired or enhanced FcRn-binding activity inan acidic pH range may be suitably obtained by altering amino acids inthe Fc region. Amino acid alterations of the Fc region that lead to suchdesired binding activity may be determined by comparing the FcRn-bindingactivity in an acidic pH range before and after amino acid alteration.Persons skilled in the art can appropriately perform amino acidalteration using known methods such as overlap extension PCR andsite-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA(1985) 82, 488-492)) similarly to the aforementioned methods used toalter antigen-binding activity.

An Fc region having FcRn-binding activity in an acidic pH range that iscontained in the antigen-binding molecules of the present invention maybe obtained by any methods, but specifically, an FcRn-binding domainhaving FcRn-binding activity or enhanced FcRn-binding activity in anacidic pH range may be obtained by altering amino acids of human IgGimmunoglobulin that is used as a starting Fc region. Examples ofpreferred IgG immunoglobulin Fc regions to be altered include the Fcregion of human IgG (IgG1, IgG2, IgG3, or IgG4, and their variants).Amino acids at any positions may be altered to other amino acids as longas the Fc region has FcRn-binding activity in an acidic pH range or itshuman FcRn-binding activity in an acidic range can be enhanced. When anantigen-binding molecule includes the Fc region of human IgG1, it ispreferred to include alterations that result in enhancement ofFcRn-binding in an acidic pH range as compared to the binding activityof the starting Fc region of human IgG1. Examples of amino acids towhich such alterations can be made preferably include, for example,amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286,288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378,380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and/or447 according to EU numbering, as described in WO 2000/042072.Similarly, examples of amino acids to which such alterations can be madealso preferably include amino acids at positions 251, 252, 254, 255,256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and/or 436according to EU numbering, as described in WO 2002/060919. Furthermore,examples of amino acids to which such alterations can be made alsoinclude amino acids at positions 250, 314, and 428 according to EUnumbering as described in WO 2004/092219. In addition, examples of aminoacids to which such alterations can be made also preferably includeamino acids at positions 251, 252, 307, 308, 378, 428, 430, 434, and/or436 according to EU numbering as described in WO 2010/045193. Alterationof these amino acids enhances the binding of an IgG immunoglobulin Fcregion to FcRn in an acidic pH range.

In the present invention, an Fc region having FcRn-binding activity in aneutral pH range is preferred. If the domain is an Fc region alreadyhaving FcRn-binding activity in a neutral pH range, it may be used as itis. When the domain has no or weak FcRn-binding activity in a neutral pHrange, an Fc region having desired FcRn-binding activity may be obtainedby altering the amino acids in the antigen-binding molecule. Also, an Fcregion having desired or enhanced FcRn-binding activity in a neutral pHrange may be suitably obtained by altering amino acids in the Fc region.Amino acid alterations of the Fc region that lead to such desiredbinding activity may be determined by comparing the FcRn-bindingactivity in a neutral pH range before and after amino acid alteration.Persons skilled in the art can suitably perform amino acid alterationusing known methods such as overlap extension PCR or site-directedmutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82,488-492)) similarly to the aforementioned methods used to alterantigen-binding activity.

An Fc region having FcRn-binding activity in a neutral pH range that iscontained in the antigen-binding molecule of the present invention maybe obtained by any method, but specifically, an FcRn-binding domainhaving FcRn-binding activity or enhanced FcRn-binding activity in aneutral pH range may be obtained by amino acid modification of human IgGimmunoglobulin used as a starting Fc region. Examples of preferred IgGimmunoglobulin Fc regions to be modified include the Fc region of humanIgG (IgG1, IgG2, IgG3, or IgG4, and their variants). Amino acids at anypositions may be altered to other amino acids as long as the Fc regionhas FcRn-binding activity in a neutral pH range or its humanFcRn-binding activity in the neutral range can be enhanced. When anantigen-binding molecule includes the Fc region of human IgG1, it ispreferred to include alterations that result in enhancement ofFcRn-binding in a neutral pH range compared to the binding activity ofthe starting Fc region of human IgG1. KD values for FcRn in a neutral pHrange are determined as described above by the method described in theJournal of Immunology (2009) 182: 7663-7671 (antigen-binding moleculesare immobilized onto a chip and human FcRn is allowed to flow as theanalyte).

Examples of preferred IgG immunoglobulin Fc regions to be alteredinclude the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and theirvariants). Amino acids at any positions may be altered to other aminoacids as long as the Fc region has FcRn-binding activity in a neutral pHrange or its human FcRn-binding activity in a neutral range can beenhanced. When an antigen-binding molecule includes the Fc region ofhuman IgG1, it is preferred to include alterations that result inenhancement of FcRn-binding in a neutral pH range compared to thebinding activity of the starting Fc region of human IgG1. In order toproduce Fc regions to which such alterations have been made, variousmutations shown in Table 5 were introduced into VH3-IgG1 (SEQ ID NO: 17)and evaluated. Variants (IgG1-F1 to IgG1-F1052) each containing aproduced heavy chain and a light chain, L (WT) (SEQ ID NO: 18), wereexpressed and purified according to the methods described in ReferenceExample 1.

Binding between an antibody and human FcRn was analyzed according to themethod described in Example 3-3. The binding activity of the variants tohuman FcRn under neutral conditions (pH7.0) measured using Biacore areshown in Table 5 (Table 5-1 to Table 5-33).

TABLE 5-1 VARIANT KD (M) AMINO ACID ALTERATION POSITION F1 8.10E−07N434W F2 3.20E−06 M252Y/S254T/T256E F3 2.50E−06 N434Y F4 5.80E−06 N434SF5 6.80E−06 N434A F7 5.60E−06 M252Y F8 4.20E−06 M252W F9 1.40E−07M252Y/S254T/T256E/N434Y F10 6.90E−08 M252Y/S254T/T256E/N434W F113.10E−07 M252Y/N434Y F12 1.70E−07 M252Y/N434W F13 3.20E−07 M252W/N434YF14 1.80E−07 M252W/N434W F19 4.60E−07 P257L/N434Y F20 4.60E−07V308F/N434Y F21 3.00E−08 M252Y/V308P/N434Y F22 2.00E−06 M428L/N434S F259.20E−09 M252Y/S254T/T256E/V308P/N434W F26 1.00E−06 I332V F27 7.40E−06G237M F29 1.40E−06 I332V/N434Y F31 2.80E−06 G237M/V308F F32 8.00E−07S254T/N434W F33 2.30E−06 S254T/N434Y F34 2.80E−07 T256E/N434W F358.40E−07 T256E/N434Y F36 3.60E−07 S254T/T256E/N434W F37 1.10E−06S254T/T256E/N434Y F38 1.00E−07 M252Y/S254T/N434W F39 3.00E−07M252Y/S254T/N434Y F40 8.20E−08 M252Y/T256E/N434W F41 1.50E−07M252Y/T256E/N434Y

TABLE 5-2 F42 1.00E−06 M252Y/S254T/T256E/N434A F43 1.70E−06 M252Y/N434AF44 1.10E−06 M252W/N434A F47 2.40E−07 M252Y/T256Q/N434W F48 3.20E−07M252Y/T256Q/N434Y F49 5.10E−07 M252F/T256D/N434W F50 1.20E−06M252F/T256D/N434Y F51 8.10E−06 N434F/Y436H F52 3.10E−06H433K/N434F/Y436H F53 1.00E−06 I332V/N434W F54 8.40E−08 V308P/N434W F569.40E−07 I332V/M428L/N434Y F57 1.10E−05 G385D/Q386P/N389S F58 7.70E−07G385D/Q386P/N389S/N434W F59 2.40E−06 G385D/Q386P/N389S/N434Y F601.10E−05 G385H F61 9.70E−07 G385H/N434W F62 1.90E−06 G385H/N434Y F632.50E−06 N434F F64 5.30E−06 N434H F65 2.90E−07 M252Y/S254T/T256E/N434FF66 4.30E−07 M252Y/S254T/T256E/N434H F67 6.30E−07 M252Y/N434F F689.30E−07 M252Y/N434H F69 5.10E−07 M428L/N434W F70 1.50E−06 M428L/N434YF71 8.30E−08 M252Y/S254T/T256E/M428L/N434W F72 2.00E−07M252Y/S254T/T256E/M428L/N434Y F73 1.70E−07 M252Y/M428L/N434W F744.60E−07 M252Y/M428L/N434Y F75 1.40E−06 M252Y/M428L/N434A F76 1.00E−06M252Y/S254T/T256E/M428L/N434A F77 9.90E−07 T256E/M428L/N434Y F787.80E−07 S254T/M428L/N434W

TABLE 5-3 F79 5.90E−06 S254T/T256E/N434A F80 2.70E−06 M252Y/T256Q/N434AF81 1.60E−06 M252Y/T256E/N434A F82 1.10E−06 T256Q/N434W F83 2.60E−06T256Q/N434Y F84 2.80E−07 M252W/T256Q/N434W F85 5.50E−07M252W/T256Q/N434Y F86 1.50E−06 S254T/T256Q/N434W F87 4.30E−06S254T/T256Q/N434Y F88 1.90E−07 M252Y/S254T/T256Q/N434W F89 3.60E−07M252Y/S254T/T256Q/N434Y F90 1.90E−08 M252Y/T256E/V308P/N434W F914.80E−08 M252Y/V308P/M428L/N434Y F92 1.10E−08M252Y/S254T/T256E/V308P/M428L/N434W F93 7.40E−07 M252W/M428L/N434W F943.70E−07 P257L/M428L/N434Y F95 2.60E−07 M252Y/S254T/T256E/M428L/N434FF99 6.20E−07 M252Y/T256E/N434H F101 1.10E−07 M252W/T256Q/P257L/N434YF103 4.40E−08 P238A/M252Y/V308P/N434Y F104 3.70E−08M252Y/D265A/V308P/N434Y F105 7.50E−08 M252Y/T307A/V308P/N434Y F1063.70E−08 M252Y/V303A/V308P/N434Y F107 3.40E−08 M252Y/V308P/D376A/N434YF108 4.10E−08 M252Y/V305A/V308P/N434Y F109 3.20E−08M252Y/V308P/Q311A/N434Y F111 3.20E−08 M252Y/V308P/K317A/N434Y F1126.40E−08 M252Y/V308P/E380A/N434Y F113 3.20E−08 M252Y/V308P/E382A/N434YF114 3.80E−08 M252Y/V308P/S424A/N434Y F115 6.60E−06 T307A/N434A F1168.70E−06 E380A/N434A F118 1.40E−05 M428L F119 5.40E−06 T250Q/M428L

TABLE 5-4 F120 6.30E−08 P257L/V308P/M428L/N434Y F121 1.50E−08M252Y/T256E/V308P/M428L/N434W F122 1.20E−07 M252Y/T256E/M428L/N434W F1233.00E−08 M252Y/T256E/V308P/N434Y F124 2.90E−07 M252Y/T256E/M428L/N434YF125 2.40E−08 M252Y/S254T/T256E/V308P/M428L/N434Y F128 1.70E−07P257L/M428L/N434W F129 2.20E−07 P257A/M428L/N434Y F131 3.00E−06P257G/M428L/N434Y F132 2.10E−07 P257I/M428L/N434Y F133 4.10E−07P257M/M428L/N434Y F134 2.70E−07 P257N/M428L/N434Y F135 7.50E−07P257S/M428L/N434Y F136 3.80E−07 P257T/M428L/N434Y F137 4.60E−07P257V/M428L/N434Y F139 1.50E−08 M252W/V308P/N434W F140 3.60E−08S239K/M252Y/V308P/N434Y F141 3.50E−08 M252Y/S298G/V308P/N434Y F1423.70E−08 M252Y/D270F/V308P/N434Y F143 2.00E−07 M252Y/V308A/N434Y F1455.30E−08 M252Y/V308F/N434Y F147 2.40E−07 M252Y/V308I/N434Y F149 1.90E−07M252Y/V308L/N434Y F150 2.00E−07 M252Y/V308M/N434Y F152 2.70E−07M252Y/V308Q/N434Y F154 1.80E−07 M252Y/V308T/N434Y F157 1.50E−07P257A/V308P/M428L/N434Y F158 5.90E−08 P257T/V308P/M428L/N434Y F1594.40E−08 P257V/V308P/M428L/N434Y F160 8.50E−07 M252W/M428I/N434Y F1621.60E−07 M252W/M428Y/N434Y F163 4.20E−07 M252W/M428F/N434Y F164 3.70E−07P238A/M252W/N434Y F165 2.90E−07 M252W/D265A/N434Y

TABLE 5-5 F166 1.50E−07 M252W/T307Q/N434Y F167 2.90E−07M252W/V303A/N434Y F168 3.20E−07 M252W/D376A/N434Y F169 2.90E−07M252W/V305A/N434Y F170 1.70E−07 M252W/Q311A/N434Y F171 1.90E−07M252W/D312A/N434Y F172 2.20E−07 M252W/K317A/N434Y F173 7.70E−07M252W/E380A/N434Y F174 3.40E−07 M252W/E382A/N434Y F175 2.70E−07M252W/S424A/N434Y F176 2.90E−07 S239K/M252W/N434Y F177 2.80E−07M252W/S298G/N434Y F178 2.70E−07 M252W/D270F/N434Y F179 3.10E−07M252W/N325G/N434Y F182 6.60E−08 P257A/M428L/N434W F183 2.20E−07P257T/M428L/N434W F184 2.70E−07 P257V/M428L/N434W F185 2.60E−07M252W/I332V/N434Y F188 3.00E−06 P257I/Q311I F189 1.90E−07M252Y/T307A/N434Y F190 1.10E−07 M252Y/T307Q/N434Y F191 1.60E−07P257L/T307A/M428L/N434Y F192 1.10E−07 P257A/T307A/M428L/N434Y F1938.50E−08 P257T/T307A/M428L/N434Y F194 1.20E−07 P257V/T307A/M428L/N434YF195 5.60E−08 P257L/T307Q/M428L/N434Y F196 3.50E−08P257A/T307Q/M428L/N434Y F197 3.30E−08 P257T/T307Q/M428L/N434Y F1984.80E−08 P257V/T307Q/M428L/N434Y F201 2.10E−07 M252Y/T307D/N434Y F2032.40E−07 M252Y/T307F/N434Y F204 2.10E−07 M252Y/T307G/N434Y F205 2.00E−07M252Y/T307H/N434Y F206 2.30E−07 M252Y/T307I/N434Y

TABLE 5-6 F207 9.40E−07 M252Y/T307K/N434Y F208 3.90E−07M252Y/T307L/N434Y F209 1.30E−07 M252Y/T307M/N434Y F210 2.90E−07M252Y/T307N/N434Y F211 2.40E−07 M252Y/T307P/N434Y F212 6.80E−07M252Y/T307R/N434Y F213 2.30E−07 M252Y/T307S/N434Y F214 1.70E−07M252Y/T307V/N434Y F215 9.60E−08 M252Y/T307W/N434Y F216 2.30E−07M252Y/T307Y/N434Y F217 2.30E−07 M252Y/K334L/N434Y F218 2.60E−07M252Y/G385H/N434Y F219 2.50E−07 M252Y/T289H/N434Y F220 2.50E−07M252Y/Q311H/N434Y F221 3.10E−07 M252Y/D312H/N434Y F222 3.40E−07M252Y/N315H/N434Y F223 2.70E−07 M252Y/K360H/N434Y F225 1.50E−06M252Y/L314R/N434Y F226 5.40E−07 M252Y/L314K/N434Y F227 1.20E−07M252Y/N286E/N434Y F228 2.30E−07 M252Y/L309E/N434Y F229 5.10E−07M252Y/R255E/N434Y F230 2.50E−07 M252Y/P387E/N434Y F236 8.90E−07K248I/M428L/N434Y F237 2.30E−07 M252Y/M428A/N434Y F238 7.40E−07M252Y/M428D/N434Y F240 7.20E−07 M252Y/M428F/N434Y F241 1.50E−06M252Y/M428G/N434Y F242 8.50E−07 M252Y/M428H/N434Y F243 1.80E−07M252Y/M428I/N434Y F244 1.30E−06 M252Y/M428K/N434Y F245 4.70E−07M252Y/M428N/N434Y F246 1.10E−06 M252Y/M428P/N434Y F247 4.40E−07M252Y/M428Q/N434Y

TABLE 5-7 F249 6.40E−07 M252Y/M428S/N434Y F250 2.90E−07M252Y/M428T/N434Y F251 1.90E−07 M252Y/M428V/N434Y F252 1.00E−06M252Y/M428W/N434Y F253 7.10E−07 M252Y/M428Y/N434Y F254 7.50E−08M252W/T307Q/M428Y/N434Y F255 1.10E−07 M252W/Q311A/M428Y/N434Y F2565.40E−08 M252W/T307Q/Q311A/M428Y/N434Y F257 5.00E−07M252Y/T307A/M428Y/N434Y F258 3.20E−07 M252Y/T307Q/M428Y/N434Y F2592.80E−07 M252Y/D270F/N434Y F260 1.30E−07 M252Y/T307A/Q311A/N434Y F2618.40E−08 M252Y/T307Q/Q311A/N434Y F262 1.90E−07 M252Y/T307A/Q311H/N434YF263 1.10E−07 M252Y/T307Q/Q311H/N434Y F264 2.80E−07 M252Y/E382A/N434YF265 6.80E−07 M252Y/E382A/M428Y/N434Y F266 4.70E−07M252Y/T307A/E382A/M428Y/N434Y F267 3.20E−07M252Y/T307Q/E382A/M428Y/N434Y F268 6.30E−07 P238A/M252Y/M428F/N434Y F2695.20E−07 M252Y/V305A/M428F/N434Y F270 6.60E−07 M252Y/N325G/M428F/N434YF271 6.90E−07 M252Y/D376A/M428F/N434Y F272 6.80E−07M252Y/E380A/M428F/N434Y F273 6.50E−07 M252Y/E382A/M428F/N434Y F2747.60E−07 M252Y/E380A/E382A/M428F/N434Y F275 4.20E−08S239K/M252Y/V308P/E382A/N434Y F276 4.10E−08M252Y/D270F/V308P/E382A/N434Y F277 1.30E−07S239K/M252Y/V308P/M428Y/N434Y F278 3.00E−08M252Y/T307Q/V308P/E382A/N434Y F279 6.10E−08M252Y/V308P/Q311H/E382A/N434Y F280 4.10E−08S239K/M252Y/D270F/V308P/N434Y F281 9.20E−08M252Y/V308P/E382A/M428F/N434Y F282 2.90E−08M252Y/V308P/E382A/M428L/N434Y

TABLE 5-8 F283 1.00E−07 M252Y/V308P/E382A/M428Y/N434Y F284 1.00E−07M252Y/V308P/M428Y/N434Y F285 9.90E−08 M252Y/V308P/M428F/N434Y F2861.20E−07 S239K/M252Y/V308P/E382A/M428Y/N434Y F287 1.00E−07M252Y/V308P/E380A/E382A/M428F/N434Y F288 1.90E−07M252Y/T256E/E382A/N434Y F289 4.80E−07 M252Y/T256E/M428Y/N434Y F2904.60E−07 M252Y/T256E/E382A/M428Y/N434Y F292 2.30E−08S239K/M252Y/V308P/E382A/M428I/N434Y F293 5.30E−08M252Y/V308P/E380A/E382A/M428I/N434Y F294 1.10E−07S239K/M252Y/V308P/M428F/N434Y F295 6.80E−07S239K/M252Y/E380A/E382A/M428F/N434Y F296 4.90E−07M252Y/Q311A/M428Y/N434Y F297 5.10E−07 M252Y/D312A/M428Y/N434Y F2984.80E−07 M252Y/Q311A/D312A/M428Y/N434Y F299 9.40E−08S239K/M252Y/V308P/Q311A/M428Y/N434Y F300 8.30E−08S239K/M252Y/V308P/D312A/M428Y/N434Y F301 7.20E−08S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y F302 1.90E−07M252Y/T256E/T307P/N434Y F303 6.70E−07 M252Y/T307P/M428Y/N434Y F3041.60E−08 M252W/V308P/M428Y/N434Y F305 2.70E−08M252Y/T256E/V308P/E382A/N434Y F306 3.60E−08 M252W/V308P/E382A/N434Y F3073.60E−08 S239K/M252W/V308P/E382A/N434Y F308 1.90E−08S239K/M252W/V308P/E382A/M428Y/N434Y F310 9.40E−08S239K/M252W/V308P/E382A/M428I/N434Y F311 2.80E−08S239K/M252W/V308P/M428F/N434Y F312 4.50E−07S239K/M252W/E380A/E382A/M428F/N434Y F313 6.50E−07S239K/M252Y/T307P/M428Y/N434Y F314 3.20E−07M252Y/T256E/Q311A/D312A/M428Y/N434Y F315 6.80E−07S239K/M252Y/M428Y/N434Y F316 7.00E−07 S239K/M252Y/D270F/M428Y/N434Y F3171.10E−07 S239K/M252Y/D270F/V308P/M428Y/N434Y F318 1.80E−08S239K/M252Y/V308P/M428I/N434Y

TABLE 5-9 F320 2.00E−08 S239K/M252Y/V308P/N325G/E382A/M428I/N434Y F3213.20E−08 S239K/M252Y/D270F/V308P/N325G/N434Y F322 9.20E−08S239K/M252Y/D270F/T307P/V308P/N434Y F323 2.70E−08S239K/M252Y/T256E/D270F/V308P/N434Y F324 2.80E−08S239K/M252Y/D270F/T307Q/V308P/N434Y F325 2.10E−08S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y F326 7.50E−08S239K/M252Y/D270F/T307Q/Q311A/N434Y F327 6.50E−08S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y F328 1.90E−08S239K/M252Y/D270F/V308P/M428I/N434Y F329 1.20E−08S239K/M252Y/D270F/N286E/V308P/N434Y F330 3.60E−08S239K/M252Y/D270F/V308P/L309E/N434Y F331 3.00E−08S239K/M252Y/D270F/V308P/P387E/N434Y F333 7.40E−08S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y F334 1.90E−08S239K/M252Y/D270F/V308P/N325G/M428I/N434Y F335 1.50E−08S239K/M252Y/T256E/D270F/V308P/M428I/N434Y F336 1.40E−08S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/ N434Y F337 5.60E−08S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y F338 7.70E−09S239K/M252Y/D270F/N286E/V308P/M428I/N434Y F339 1.90E−08S239K/M252Y/D270F/V308P/L309E/M428I/N434Y F343 3.20E−08S239K/M252Y/D270F/V308P/M428L/N434Y F344 3.00E−08S239K/M252Y/V308P/M428L/N434Y F349 1.50E−07S239K/M252Y/V308P/L309P/M428L/N434Y F350 1.70E−07S239K/M252Y/V308P/L309R/M428L/N434Y F352 6.00E−07S239K/M252Y/L309P/M428L/N434Y F353 1.10E−06S239K/M252Y/L309R/M428L/N434Y F354 2.80E−08S239K/M252Y/T307Q/V308P/M428L/N434Y F356 3.40E−08S239K/M252Y/D270F/V308P/L309E/P387E/N434Y F357 1.60E−08S239K/M252Y/T256E/D270F/V308P/N325G/M428I/ N434Y F358 1.00E−07S239K/M252Y/T307Q/N434Y F359 4.20E−07 P257V/T307Q/M428I/N434Y F3601.30E−06 P257V/T307Q/M428V/N434Y F362 5.40E−08P257V/T307Q/N325G/M428L/N434Y F363 4.10E−08P257V/T307Q/Q311A/M428L/N434Y F364 3.50E−08P257V/T307Q/Q311A/N325G/M428L/N434Y

TABLE 5-10 F365 5.10E−08 P257V/V305A/T307Q/M428L/N434Y F367 1.50E−08S239K/M252Y/E258H/D270F/T307Q/V308P/Q311A/N434Y F368 2.00E−08S239K/M252Y/D270F/V308P/N325G/E382A/M428I/N434Y F369 7.50E−08M252Y/P257V/T307Q/M428I/N434Y F372 1.30E−08S239K/M252W/V308P/M428Y/N434Y F373 1.10E−08S239K/M252W/V308P/Q311A/M428Y/N434Y F374 1.20E−08S239K/M252W/T256E/V308P/M428Y/N434Y F375 5.50E−09S239K/M252W/N286E/V308P/M428Y/N434Y F376 9.60E−09S239K/M252Y/T256E/D270F/N286E/V308P/N434Y F377 1.30E−07S239K/M252W/T307P/M428Y/N434Y F379 9.00E−09S239K/M252W/T256E/V308P/Q311A/M428Y/N434Y F380 5.60E−09S239K/M252W/T256E/N286E/V308P/M428Y/N434Y F381 1.10E−07P257V/T307A/Q311A/M428L/N434Y F382 8.70E−08P257V/V305A/T307A/M428L/N434Y F386 3.20E−08 M252Y/V308P/L309E/N434Y F3871.50E−07 M252Y/V308P/L309D/N434Y F388 7.00E−08 M252Y/V308P/L309A/N434YF389 1.70E−08 M252W/V308P/L309E/M428Y/N434Y F390 6.80E−08M252W/V308P/L309D/M428Y/N434Y F391 3.60E−08M252W/V308P/L309A/M428Y/N434Y F392 6.90E−09S239K/M252Y/N286E/V308P/M428I/N434Y F393 1.20E−08S239K/M252Y/N286E/V308P/N434Y F394 5.30E−08S239K/M252Y/T307Q/Q311A/M428I/N434Y F395 2.40E−08S239K/M252Y/T256E/V308P/N434Y F396 2.00E−08S239K/M252Y/D270F/N286E/T307Q/Q311A/M428I/N434Y F397 4.50E−08S239K/M252Y/D270F/T307Q/Q311A/P387E/M428I/N434Y F398 4.40E−09S239K/M252Y/D270F/N286E/T307Q/V308P/Q311A/M428I/N434Y F399 6.50E−09S239K/M252Y/D270F/N286E/T307Q/V308P/M428I/N434Y F400 6.10E−09S239K/M252Y/D270F/N286E/V308P/Q311A/M428I/N434Y F401 6.90E−09S239K/M252Y/D270F/N286E/V308P/P387E/M428I/N434Y F402 2.30E−08P257V/T307Q/M428L/N434W F403 5.10E−08 P257V/T307A/M428L/N434W F4049.40E−08 P257A/T307Q/L309P/M428L/N434Y F405 1.70E−07P257V/T307Q/L309P/M428L/N434Y

TABLE 5-11 F406 1.50E−07 P257A/T307Q/L309R/M428L/N434Y F407 1.60E−07P257V/T307Q/L309R/M428L/N434Y F408 2.50E−07 P257V/N286E/M428L/N434Y F4092.00E−07 P257V/P387E/M428L/N434Y F410 2.20E−07 P257V/T307H/M428L/N434YF411 1.30E−07 P257V/T307N/M428L/N434Y F412 8.80E−08P257V/T307G/M428L/N434Y F413 1.20E−07 P257V/T307P/M428L/N434Y F4141.10E−07 P257V/T307S/M428L/N434Y F415 5.60E−08P257V/N286E/T307A/M428L/N434Y F416 9.40E−08P257V/T307A/P387E/M428L/N434Y F418 6.20E−07S239K/M252Y/T307P/N325G/M428Y/N434Y F419 1.60E−07M252Y/T307A/Q311H/K360H/N434Y F420 1.50E−07M252Y/T307A/Q311H/P387E/N434Y F421 1.30E−07M252Y/T307A/Q311H/M428A/N434Y F422 1.80E−07M252Y/T307A/Q311H/E382A/N434Y F423 8.40E−08 M252Y/T307W/Q311H/N434Y F4249.40E−08 S239K/P257A/V308P/M428L/N434Y F425 8.00E−08P257A/V308P/L309E/M428L/N434Y F426 8.40E−08 P257V/T307Q/N434Y F4271.10E−07 M252Y/P257V/T307Q/M428V/N434Y F428 8.00E−08M252Y/P257V/T307Q/M428L/N434Y F429 3.70E−08 M252Y/P257V/T307Q/N434Y F4308.10E−08 M252Y/P257V/T307Q/M428Y/N434Y F431 6.50E−08M252Y/P257V/T307Q/M428F/N434Y F432 9.20E−07P257V/T307Q/Q311A/N325G/M428V/N434Y F433 6.00E−08P257V/T307Q/Q311A/N325G/N434Y F434 2.00E−08P257V/T307Q/Q311A/N325G/M428Y/N434Y F435 2.50E−08P257V/T307Q/Q311A/N325G/M428F/N434Y F436 2.50E−07P257A/T307Q/M428V/N434Y F437 5.70E−08 P257A/T307Q/N434Y F438 3.60E−08P257A/T307Q/M428Y/N434Y F439 4.00E−08 P257A/T307Q/M428F/N434Y F4401.50E−08 P257V/N286E/T307Q/Q311A/N325G/M428L/N434Y

TABLE 5-12 F441 1.80E−07 P257A/Q311A/M428L/N434Y F442 2.00E−07P257A/Q311H/M428L/N434Y F443 5.50E−08 P257A/T307Q/Q311A/M428L/N434Y F4441.40E−07 P257A/T307A/Q311A/M428L/N434Y F445 6.20E−08P257A/T307Q/Q311H/M428L/N434Y F446 1.10E−07P257A/T307A/Q311H/M428L/N434Y F447 1.40E−08P257A/N286E/T307Q/M428L/N434Y F448 5.30E−08P257A/N286E/T307A/M428L/N434Y F449 5.70E−07S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y F450 5.20E−07S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y F451 1.00E−07P257S/T307A/M428L/N434Y F452 1.40E−07 P257M/T307A/M428L/N434Y F4537.80E−08 P257N/T307A/M428L/N434Y F454 9.60E−08 P257I/T307A/M428L/N434YF455 2.70E−08 P257V/T307Q/M428Y/N434Y F456 3.40E−08P257V/T307Q/M428F/N434Y F457 4.00E−08 S239K/P257V/V308P/M428L/N434Y F4581.50E−08 P257V/T307Q/V308P/N325G/M428L/N434Y F459 1.30E−08P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y F460 4.70E−08P257V/T307A/V308P/N325G/M428L/N434Y F462 8.50E−08P257A/V308P/N325G/M428L/N434Y F463 1.30E−07P257A/T307A/V308P/M428L/N434Y F464 5.50E−08P257A/T307Q/V308P/M428L/N434Y F465 2.10E−08P257V/N286E/T307Q/N325G/M428L/N434Y F466 3.50E−07 T256E/P257V/N434Y F4675.70E−07 T256E/P257T/N434Y F468 5.70E−08 S239K/P257T/V308P/M428L/N434YF469 5.60E−08 P257T/V308P/N325G/M428L/N434Y F470 5.40E−08T256E/P257T/V308P/N325G/M428L/N434Y F471 6.60E−08P257T/V308P/N325G/E382A/M428L/N434Y F472 5.40E−08P257T/V308P/N325G/P387E/M428L/N434Y F473 4.50E−07P257T/V308P/L309P/N325G/M428L/N434Y F474 3.50E−07P257T/V308P/L309R/N325G/M428L/N434Y F475 4.30E−08T256E/P257V/T307Q/M428L/N434Y

TABLE 5-13 F476 5.50E−08 P257V/T307Q/E382A/M428L/N434Y F477 4.30E−08P257V/T307Q/P387E/M428L/N434Y F480 3.90E−08 P257L/V308P/N434Y F4815.60E−08 P257T/T307Q/N434Y F482 7.00E−08 P257V/T307Q/N325G/N434Y F4835.70E−08 P257V/T307Q/Q311A/N434Y F484 6.20E−08 P257V/V305A/T307Q/N434YF485 9.70E−08 P257V/N286E/T307A/N434Y F486 3.40E−07P257V/T307Q/L309R/Q311H/M428L/N434Y F488 3.50E−08P257V/V308P/N325G/M428L/N434Y F490 7.50E−08S239K/P257V/V308P/Q311H/M428L/N434Y F492 9.80E−08P257V/V305A/T307A/N325G/M428L/N434Y F493 4.90E−07S239K/D270F/T307P/N325G/M428Y/N434Y F497 3.10E−06P257T/T307A/M428V/N434Y F498 1.30E−06 P257A/M428V/N434Y F499 5.20E−07P257A/T307A/M428V/N434Y F500 4.30E−08 P257S/T307Q/M428L/N434Y F5061.90E−07 P257V/N297A/T307Q/M428L/N434Y F507 5.10E−08P257V/N286A/T307Q/M428L/N434Y F508 1.10E−07P257V/T307Q/N315A/M428L/N434Y F509 5.80E−08P257V/T307Q/N384A/M428L/N434Y F510 5.30E−08P257V/T307Q/N389A/M428L/N434Y F511 4.20E−07 P257V/N434Y F512 5.80E−07P257T/N434Y F517 3.10E−07 P257V/N286E/N434Y F518 4.20E−07P257T/N286E/N434Y F519 2.60E−08 P257V/N286E/T307Q/N434Y F521 1.10E−08P257V/N286E/T307Q/M428Y/N434Y F523 2.60E−08P257V/V305A/T307Q/M428Y/N434Y F526 1.90E−08 P257T/T307Q/M428Y/N434Y F5279.40E−09 P257V/T307Q/V308P/N325G/M428Y/N434Y F529 2.50E−08P257T/T307Q/M428F/N434Y F533 1.20E−08 P257A/N286E/T307Q/M428F/N434Y F5341.20E−08 P257A/N286E/T307Q/M428Y/N434Y

TABLE 5-14 F535 3.90E−08 T250A/P257V/T307Q/M428L/N434Y F538 9.90E−08T250F/P257V/T307Q/M428L/N434Y F541 6.00E−08T250I/P257V/T307Q/M428L/N434Y F544 3.10E−08T250M/P257V/T307Q/M428L/N434Y F549 5.40E−08T250S/P257V/T307Q/M428L/N434Y F550 5.90E−08T250V/P257V/T307Q/M428L/N434Y F551 1.20E−07T250W/P257V/T307Q/M428L/N434Y F552 1.10E−07T250Y/P257V/T307Q/M428L/N434Y F553 1.70E−07 M252Y/Q311A/N434Y F5542.80E−08 S239K/M252Y/S254T/V308P/N434Y F556 1.50E−06 M252Y/T307Q/Q311AF559 8.00E−08 M252Y/S254T/N286E/N434Y F560 2.80E−08M252Y/S254T/V308P/N434Y F561 1.40E−07 M252Y/S254T/T307A/N434Y F5628.30E−08 M252Y/S254T/T307Q/N434Y F563 1.30E−07 M252Y/S254T/Q311A/N434YF564 1.90E−07 M252Y/S254T/Q311H/N434Y F565 9.20E−08M252Y/S254T/T307A/Q311A/N434Y F566 6.10E−08M252Y/S254T/T307Q/Q311A/N434Y F567 2.20E−07 M252Y/S254T/M428I/N434Y F5681.10E−07 M252Y/T256E/T307A/Q311H/N434Y F569 2.00E−07M252Y/T256Q/T307A/Q311H/N434Y F570 1.30E−07M252Y/S254T/T307A/Q311H/N434Y F571 8.10E−08M252Y/N286E/T307A/Q311H/N434Y F572 1.00E−07M252Y/T307A/Q311H/M428I/N434Y F576 1.60E−06 M252Y/T256E/T307Q/Q311H F5771.30E−06 M252Y/N286E/T307A/Q311A F578 5.70E−07 M252Y/N286E/T307Q/Q311AF580 8.60E−07 M252Y/N286E/T307Q/Q311H F581 7.20E−08M252Y/T256E/N286E/N434Y F582 7.50E−07 S239K/M252Y/V308P F583 7.80E−07S239K/M252Y/V308P/E382A F584 6.30E−07 S239K/M252Y/T256E/V308P F5852.90E−07 S239K/M252Y/N286E/V308P

TABLE 5-15 F586 1.40E−07 S239K/M252Y/N286E/V308P/M428I F587 1.90E−07M252Y/N286E/M428L/N434Y F592 2.00E−07 M252Y/S254T/E382A/N434Y F5933.10E−08 S239K/M252Y/S254T/V308P/M428I/N434Y F594 1.60E−08S239K/M252Y/T256E/V308P/M428I/N434Y F595 1.80E−07S239K/M252Y/M428I/N434Y F596 4.00E−07 M252Y/D312A/E382A/M428Y/N434Y F5972.20E−07 M252Y/E382A/P387E/N434Y F598 1.40E−07 M252Y/D312A/P387E/N434YF599 5.20E−07 M252Y/P387E/M428Y/N434Y F600 2.80E−07M252Y/T256Q/E382A/N434Y F601 9.60E−09 M252Y/N286E/V308P/N434Y F608G236A/S239D/I332E F611 2.80E−07 M252Y/V305T/T307P/V308I/L309A/N434Y F6123.60E−07 M252Y/T307P/V308I/L309A/N434Y F613 S239D/A330L/I332E F616S239D/K326D/L328Y F617 7.40E−07 S239K/N434W F618 6.40E−07S239K/V308F/N434Y F619 3.10E−07 S239K/M252Y/N434Y F620 2.10E−07S239K/M252Y/S254T/N434Y F621 1.50E−07 S239K/M252Y/T307A/Q311H/N434Y F6223.50E−07 S239K/M252Y/T256Q/N434Y F623 1.80E−07 S239K/M252W/N434W F6241.40E−08 S239K/P257A/N286E/T307Q/M428L/N434Y F625 7.60E−08S239K/P257A/T307Q/M428L/N434Y F626 1.30E−06 V308P F629 3.90E−08M252Y/V279L/V308P/N434Y F630 3.70E−08 S239K/M252Y/V279L/V308P/N434Y F6332.40E−08 M252Y/V282D/V308P/N434Y F634 3.20E−08S239K/M252Y/V282D/V308P/N434Y F635 4.50E−08 M252Y/V284K/V308P/N434Y F6364.80E−08 S239K/M252Y/V284K/V308P/N434Y F637 1.50E−07M252Y/K288S/V308P/N434Y

TABLE 5-16 F638 1.40E−07 S239K/M252Y/K288S/V308P/N434Y F639 2.70E−08M252Y/V308P/G385R/N434Y F640 3.60E−08 S239K/M252Y/V308P/G385R/N434Y F6413.00E−08 M252Y/V308P/Q386K/N434Y F642 3.00E−08S239K/M252Y/V308P/Q386K/N434Y F643 3.20E−08L235G/G236R/S239K/M252Y/V308P/N434Y F644 3.00E−08G236R/S239K/M252Y/V308P/N434Y F645 3.30E−08S239K/M252Y/V308P/L328R/N434Y F646 3.80E−08S239K/M252Y/N297A/V308P/N434Y F647 2.90E−08 P238D/M252Y/V308P/N434Y F648P238D F649 1.20E−07 S239K/M252Y/N286E/N434Y F650 1.70E−07S239K/M252Y/T256E/N434Y F651 1.80E−07 S239K/M252Y/Q311A/N434Y F6522.40E−07 P238D/M252Y/N434Y F654 3.20E−08 L235K/S239K/M252Y/V308P/N434YF655 3.40E−08 L235R/S239K/M252Y/V308P/N434Y F656 3.30E−08G237K/S239K/M252Y/V308P/N434Y F657 3.20E−08G237R/S239K/M252Y/V308P/N434Y F658 3.20E−08P238K/S239K/M252Y/V308P/N434Y F659 3.00E−08P238R/S239K/M252Y/V308P/N434Y F660 3.10E−08S239K/M252Y/V308P/P329K/N434Y F661 3.40E−08S239K/M252Y/V308P/P329R/N434Y F663 6.40E−09S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y F664 3.90E−08M252Y/N286A/V308P/N434Y F665 2.00E−08 M252Y/N286D/V308P/N434Y F6662.10E−08 M252Y/N286F/V308P/N434Y F667 3.00E−08 M252Y/N286G/V308P/N434YF668 4.00E−08 M252Y/N286H/V308P/N434Y F669 3.50E−08M252Y/N286I/V308P/N434Y F670 2.10E−07 M252Y/N286K/V308P/N434Y F6712.20E−08 M252Y/N286L/V308P/N434Y F672 2.40E−08 M252Y/N286M/V308P/N434YF673 2.30E−08 M252Y/N286P/V308P/N434Y

TABLE 5-17 F674 3.20E−08 M252Y/N286Q/V308P/N434Y F675 5.10E−08M252Y/N286R/V308P/N434Y F676 3.20E−08 M252Y/N286S/V308P/N434Y F6774.70E−08 M252Y/N286T/V308P/N434Y F678 3.30E−08 M252Y/N286V/V308P/N434YF679 1.70E−08 M252Y/N286W/V308P/N434Y F680 1.50E−08M252Y/N286Y/V308P/N434Y F681 4.90E−08 M252Y/K288A/V308P/N434Y F6828.20E−08 M252Y/K288D/V308P/N434Y F683 5.00E−08 M252Y/K288E/V308P/N434YF684 5.10E−08 M252Y/K288F/V308P/N434Y F685 5.30E−08M252Y/K288G/V308P/N434Y F686 4.60E−08 M252Y/K288H/V308P/N434Y F6874.90E−08 M252Y/K288I/V308P/N434Y F688 2.80E−08 M252Y/K288L/V308P/N434YF689 4.10E−08 M252Y/K288M/V308P/N434Y F690 1.00E−07M252Y/K288N/V308P/N434Y F691 3.20E−07 M252Y/K288P/V308P/N434Y F6923.90E−08 M252Y/K288Q/V308P/N434Y F693 3.60E−08 M252Y/K288R/V308P/N434YF694 4.70E−08 M252Y/K288V/V308P/N434Y F695 4.00E−08M252Y/K288W/V308P/N434Y F696 4.40E−08 M252Y/K288Y/V308P/N434Y F6973.10E−08 S239K/M252Y/V308P/N325G/N434Y F698 2.20E−08M252Y/N286E/T307Q/Q311A/N434Y F699 2.30E−08S239K/M252Y/N286E/T307Q/Q311A/N434Y F700 5.20E−08M252Y/V308P/L328E/N434Y F705 7.10E−09 M252Y/N286E/V308P/M428I/N434Y F7061.80E−08 M252Y/N286E/T307Q/Q311A/M428I/N434Y F707 5.90E−09M252Y/N286E/T307Q/V308P/Q311A/N434Y F708 4.10E−09M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y F709 2.00E−08S239K/M252Y/N286E/T307Q/Q311A/M428I/N434Y F710 1.50E−08P238D/M252Y/N286E/T307Q/Q311A/M428I/N434Y F711 6.50E−08S239K/M252Y/T307Q/Q311A/N434Y

TABLE 5-18 F712 6.00E−08 P238D/M252Y/T307Q/Q311A/N434Y F713 2.00E−08P238D/M252Y/N286E/T307Q/Q311A/N434Y F714 2.30E−07P238D/M252Y/N325S/N434Y F715 2.30E−07 P238D/M252Y/N325M/N434Y F7162.70E−07 P238D/M252Y/N325L/N434Y F717 2.60E−07 P238D/M252Y/N325I/N434YF718 2.80E−07 P238D/M252Y/Q295M/N434Y F719 7.40E−08P238D/M252Y/N325G/N434Y F720 2.40E−08 M252Y/T307Q/V308P/Q311A/N434Y F7211.50E−08 M252Y/T307Q/V308P/Q311A/M428I/N434Y F722 2.70E−07P238D/M252Y/A327G/N434Y F723 2.80E−07 P238D/M252Y/L328D/N434Y F7242.50E−07 P238D/M252Y/L328E/N434Y F725 4.20E−08L235K/G237R/S239K/M252Y/V308P/N434Y F726 3.70E−08L235K/P238K/S239K/M252Y/V308P/N434Y F729 9.20E−07 T307A/Q311A/N434Y F7306.00E−07 T307Q/Q311A/N434Y F731 8.50E−07 T307A/Q311H/N434Y F732 6.80E−07T307Q/Q311H/N434Y F733 3.20E−07 M252Y/L328E/N434Y F734 3.10E−07G236D/M252Y/L328E/N434Y F736 3.10E−07 M252Y/S267M/L328E/N434Y F7373.10E−07 M252Y/S267L/L328E/N434Y F738 3.50E−07 P238D/M252Y/T307P/N434YF739 2.20E−07 M252Y/T307P/Q311A/N434Y F740 2.90E−07M252Y/T307P/Q311H/N434Y F741 3.10E−07 P238D/T250A/M252Y/N434Y F7449.90E−07 P238D/T250F/M252Y/N434Y F745 6.60E−07 P238D/T250G/M252Y/N434YF746 6.00E−07 P238D/T250H/M252Y/N434Y F747 2.80E−07P238D/T250I/M252Y/N434Y F749 5.10E−07 P238D/T250L/M252Y/N434Y F7503.00E−07 P238D/T250M/M252Y/N434Y F751 5.30E−07 P238D/T250N/M252Y/N434Y

TABLE 5-19 F753 1.80E−07 P238D/T250Q/M252Y/N434Y F755 3.50E−07P238D/T250S/M252Y/N434Y F756 3.70E−07 P238D/T250V/M252Y/N434Y F7571.20E−06 P238D/T250W/M252Y/N434Y F758 1.40E−06 P238D/T250Y/M252Y/N434YF759 L235K/S239K F760 L235R/S239K F761 1.10E−06 P238D/N434Y F7623.60E−08 L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y F763 3.50E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y F764 6.30E−07P238D/T307Q/Q311A/N434Y F765 8.50E−08P238D/M252Y/T307Q/L309E/Q311A/N434Y F766 6.00E−07T307A/L309E/Q311A/N434Y F767 4.30E−07 T307Q/L309E/Q311A/N434Y F7686.40E−07 T307A/L309E/Q311H/N434Y F769 4.60E−07 T307Q/L309E/Q311H/N434YF770 3.00E−07 M252Y/T256A/N434Y F771 4.00E−07 M252Y/E272A/N434Y F7723.80E−07 M252Y/K274A/N434Y F773 3.90E−07 M252Y/V282A/N434Y F774 4.00E−07M252Y/N286A/N434Y F775 6.20E−07 M252Y/K338A/N434Y F776 3.90E−07M252Y/K340A/N434Y F777 3.90E−07 M252Y/E345A/N434Y F779 3.90E−07M252Y/N361A/N434Y F780 3.90E−07 M252Y/Q362A/N434Y F781 3.70E−07M252Y/S375A/N434Y F782 3.50E−07 M252Y/Y391A/N434Y F783 4.00E−07M252Y/D413A/N434Y F784 5.00E−07 M252Y/L309A/N434Y F785 7.40E−07M252Y/L309H/N434Y F786 2.80E−08 M252Y/S254T/N286E/T307Q/Q311A/N434Y F7878.80E−08 M252Y/S254T/T307Q/L309E/Q311A/N434Y F788 4.10E−07M252Y/N315A/N434Y

TABLE 5-20 F789 1.50E−07 M252Y/N315D/N434Y F790 2.70E−07M252Y/N315E/N434Y F791 4.40E−07 M252Y/N315F/N434Y F792 4.40E−07M252Y/N315G/N434Y F793 3.30E−07 M252Y/N315I/N434Y F794 4.10E−07M252Y/N315K/N434Y F795 3.10E−07 M252Y/N315L/N434Y F796 3.40E−07M252Y/N315M/N434Y F798 3.50E−07 M252Y/N315Q/N434Y F799 4.10E−07M252Y/N315R/N434Y F800 3.80E−07 M252Y/N315S/N434Y F801 4.40E−07M252Y/N315T/N434Y F802 3.30E−07 M252Y/N315V/N434Y F803 3.60E−07M252Y/N315W/N434Y F804 4.00E−07 M252Y/N315Y/N434Y F805 3.00E−07M252Y/N325A/N434Y F806 3.10E−07 M252Y/N384A/N434Y F807 3.20E−07M252Y/N389A/N434Y F808 3.20E−07 M252Y/N389A/N390A/N434Y F809 2.20E−07M252Y/S254T/T256S/N434Y F810 2.20E−07 M252Y/A378V/N434Y F811 4.90E−07M252Y/E380S/N434Y F812 2.70E−07 M252Y/E382V/N434Y F813 2.80E−07M252Y/S424E/N434Y F814 1.20E−07 M252Y/N434Y/Y436I F815 5.50E−07M252Y/N434Y/T437R F816 3.60E−07 P238D/T250V/M252Y/T307P/N434Y F8179.80E−08 P238D/T250V/M252Y/T307Q/Q311A/N434Y F819 1.40E−07P238D/M252Y/N286E/N434Y F820 3.40E−07 L235K/S239K/M252Y/N434Y F8213.10E−07 L235R/S239K/M252Y/N434Y F822 1.10E−06P238D/T250Y/M252Y/W313Y/N434Y F823 1.10E−06P238D/T250Y/M252Y/W313F/N434Y F828 2.50E−06P238D/T250V/M252Y/I253V/N434Y

TABLE 5-21 F831 1.60E−06 P238D/T250V/M252Y/R255A/N434Y F832 2.60E−06P238D/T250V/M252Y/R255D/N434Y F833 8.00E−07P238D/T250V/M252Y/R255E/N434Y F834 8.10E−07P238D/T250V/M252Y/R255F/N434Y F836 5.00E−07P238D/T250V/M252Y/R255H/N434Y F837 5.60E−07P238D/T250V/M252Y/R255I/N434Y F838 4.30E−07P238D/T250V/M252Y/R255K/N434Y F839 3.40E−07P238D/T250V/M252Y/R255L/N434Y F840 4.20E−07P238D/T250V/M252Y/R255M/N434Y F841 1.10E−06P238D/T250V/M252Y/R255N/N434Y F843 6.60E−07P238D/T250V/M252Y/R255Q/N434Y F844 1.30E−06P238D/T250V/M252Y/R255S/N434Y F847 3.40E−07P238D/T250V/M252Y/R255W/N434Y F848 8.30E−07P238D/T250V/M252Y/R255Y/N434Y F849 3.30E−07 M252Y/D280A/N434Y F8502.90E−07 M252Y/D280E/N434Y F852 3.30E−07 M252Y/D280G/N434Y F853 3.20E−07M252Y/D280H/N434Y F855 3.20E−07 M252Y/D280K/N434Y F858 3.20E−07M252Y/D280N/N434Y F860 3.30E−07 M252Y/D280Q/N434Y F861 3.20E−07M252Y/D280R/N434Y F862 3.00E−07 M252Y/D280S/N434Y F863 2.70E−07M252Y/D280T/N434Y F867 2.80E−07 M252Y/N384A/N389A/N434Y F868 2.00E−08G236A/S239D/M252Y/N286E/T307Q/Q311A/N434Y F869 G236A/S239D F870 7.30E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y F871 7.10E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y F872 1.30E−07L235K/S239K/M252Y/N286E/N434Y F873 1.20E−07L235R/S239K/M252Y/N286E/N434Y F875 4.80E−07 M252Y/N434Y/Y436A F8778.30E−07 M252Y/N434Y/Y436E F878 1.90E−07 M252Y/N434Y/Y436F

TABLE 5-22 F879 9.20E−07 M252Y/N434Y/Y436G F880 3.90E−07M252Y/N434Y/Y436H F881 3.10E−07 M252Y/N434Y/Y436K F882 1.30E−07M252Y/N434Y/Y436L F883 2.10E−07 M252Y/N434Y/Y436M F884 4.00E−07M252Y/N434Y/Y436N F888 4.80E−07 M252Y/N434Y/Y436S F889 2.20E−07M252Y/N434Y/Y436T F890 1.10E−07 M252Y/N434Y/Y436V F891 1.70E−07M252Y/N434Y/Y436W F892 7.10E−08 M252Y/S254T/N434Y/Y436I F893 9.80E−08L235K/S239K/M252Y/N434Y/Y436I F894 9.20E−08L235R/S239K/M252Y/N434Y/Y436I F895 2.10E−08L235K/S239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F896 2.00E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F897 9.70E−08M252Y/N315D/N384A/N389A/N434Y F898 1.70E−07M252Y/N315E/N384A/N389A/N434Y F899 1.10E−07 M252Y/N315D/G316A/N434Y F9001.70E−07 M252Y/N315D/G316D/N434Y F901 1.30E−07 M252Y/N315D/G316E/N434YF902 2.20E−07 M252Y/N315D/G316F/N434Y F903 2.30E−07M252Y/N315D/G316H/N434Y F904 1.00E−07 M252Y/N315D/G316I/N434Y F9051.30E−07 M252Y/N315D/G316K/N434Y F906 1.50E−07 M252Y/N315D/G316L/N434YF907 1.30E−07 M252Y/N315D/G316M/N434Y F908 1.50E−07M252Y/N315D/G316N/N434Y F909 1.30E−07 M252Y/N315D/G316P/N434Y F9101.40E−07 M252Y/N315D/G316Q/N434Y F911 1.30E−07 M252Y/N315D/G316R/N434YF912 1.20E−07 M252Y/N315D/G316S/N434Y F913 1.10E−07M252Y/N315D/G316T/N434Y F914 1.50E−07 M252Y/N315D/G316V/N434Y F9152.30E−07 M252Y/N315D/G316W/N434Y

TABLE 5-23 F917 2.50E−07 M252Y/N286S/N434Y F918 2.80E−07M252Y/D280E/N384A/N389A/N434Y F919 3.30E−07M252Y/D280G/N384A/N389A/N434Y F920 2.50E−07M252Y/N286S/N384A/N389A/N434Y F921 1.20E−07M252Y/N286E/N384A/N389A/N434Y F922 5.90E−08L235K/S239K/M252Y/N286E/N434Y/Y436I F923 6.00E−08L235R/S239K/M252Y/N286E/N434Y/Y436I F924 3.40E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F925 3.20E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F926 1.10E−07L235K/S239K/M252Y/S254T/N434Y/Y436I F927 1.00E−07L235R/S239K/M252Y/S254T/N434Y/Y436I F928 2.90E−08M252Y/T307Q/Q311A/N434Y/Y436I F929 2.90E−08M252Y/S254T/T307Q/Q311A/N434Y/Y436I F930 1.40E−07P238D/T250V/M252Y/N286E/N434Y F931 1.20E−07P238D/T250V/M252Y/N434Y/Y436I F932 3.20E−07 T250V/M252Y/N434Y F9333.00E−07 L234R/P238D/T250V/M252Y/N434Y F934 3.10E−07G236K/P238D/T250V/M252Y/N434Y F935 3.20E−07G237K/P238D/T250V/M252Y/N434Y F936 3.20E−07G237R/P238D/T250V/M252Y/N434Y F937 3.10E−07P238D/S239K/T250V/M252Y/N434Y F938 1.60E−07L235K/S239K/M252Y/N434Y/Y436V F939 1.50E−07L235R/S239K/M252Y/N434Y/Y436V F940 1.50E−07P238D/T250V/M252Y/N434Y/Y436V F941 1.20E−08M252Y/N286E/T307Q/Q311A/N434Y/Y436V F942 4.20E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F943 4.00E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F944 1.70E−07T250V/M252Y/N434Y/Y436V F945 1.70E−08 T250V/M252Y/V308P/N434Y/Y436V F9464.30E−08 T250V/M252Y/T307Q/Q311A/N434Y/Y436V F947 1.10E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F954 5.30E−07M252Y/N434Y/H435K/Y436V F957 7.70E−07 M252Y/N434Y/H435N/Y436V F9608.00E−07 M252Y/N434Y/H435R/Y436V

TABLE 5-24 F966 3.10E−07 M252Y/S254A/N434Y F970 2.50E−06M252Y/S254G/N434Y F971 2.60E−06 M252Y/S254H/N434Y F972 2.60E−07M252Y/S254I/N434Y F978 1.30E−06 M252Y/S254Q/N434Y F980 1.80E−07M252Y/S254V/N434Y F987 4.00E−08P238D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F988 6.90E−08P238D/T250V/M252Y/N286E/N434Y/Y436V F989 1.40E−08L235R/S239K/M252Y/V308P/N434Y/Y436V F990 9.40E−09L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F991 1.30E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F992 5.10E−08L235R/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F993 3.80E−08M252Y/T307Q/Q311A/N434Y/Y436V F994 2.80E−07 M252Y/N325G/N434Y F9952.90E−07 L235R/P238D/S239K/M252Y/N434Y F996 1.30E−07L235R/P238D/S239K/M252Y/N434Y/Y436V F997 3.80E−07K248I/T250V/M252Y/N434Y/Y436V F998 8.50E−07K248Y/T250V/M252Y/N434Y/Y436V F999 2.10E−07T250V/M252Y/E258H/N434Y/Y436V F1005 N325G F1008 1.70E−07L235R/S239K/T250V/M252Y/N434Y/Y436V F1009 1.20E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1010 1.90E−07L235R/S239K/M252Y/T307A/Q311H/N434Y F1011 4.50E−08T250V/M252Y/V308P/N434Y F1012 4.70E−08L235R/S239K/T250V/M252Y/V308P/N434Y F1013 3.00E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y F1014 3.20E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1015 2.20E−08L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y F1016 3.80E−09T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F1017 4.20E−09L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10183.20E−09 L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10193.40E−07 P238D/T250V/M252Y/N325G/N434Y F1020 8.50E−08P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y

TABLE 5-25 F1021 3.30E−07 P238D/T250V/M252Y/N325A/N434Y F1022K326D/L328Y F1023 4.40E−08 S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436VF1024 4.00E−08 T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V F10253.60E−08 S239D/T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V F10268.40E−08 M252Y/T307A/Q311H/N434Y/Y436V F1027 8.60E−08L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1028 4.60E−08G236A/S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F1029 5.10E−08T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V F1030 I332E F1031 5.30E−08G236A/S239D/T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V F1032 4.30E−08P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y/Y436V F1033 1.00E−06P238D/N434W F1034 1.50E−08 L235K/S239K/M252Y/V308P/N434Y/Y436V F10351.00E−08 L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1036 1.40E−08L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F1037 6.10E−08L235K/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F1038 2.80E−07L235K/P238D/S239K/M252Y/N434Y F1039 1.30E−07L235K/P238D/S239K/M252Y/N434Y/Y436V F1040 2.00E−07L235K/S239K/T250V/M252Y/N434Y/Y436V F1041 1.40E−08L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1042 2.00E−07L235K/S239K/M252Y/T307A/Q311H/N434Y F1043 5.20E−08L235K/S239K/T250V/M252Y/V308P/N434Y F1044 3.50E−08L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1045 2.50E−08L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y F1046 4.50E−09L235K/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10473.40E−09 L235K/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10489.90E−08 L235K/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1050 3.50E−09T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V F1051 3.90E−09L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/ Y436V F10523.20E−09 L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V

TABLE 5-26 F1053 4.23E−08L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F1058 1.31E−07M252Y/Q386E/N434Y/Y436V F1059 1.39E−07 M252Y/Q386R/N434Y/Y436V F10601.43E−07 M252Y/Q386S/N434Y/Y436V F1061 1.19E−07 M252Y/P387E/N434Y/Y436VF1062 1.20E−07 M252Y/P387R/N434Y/Y436V F1063 1.43E−07M252Y/P387S/N434Y/Y436V F1064 1.32E−07 M252Y/V422E/N434Y/Y436V F10651.38E−07 M252Y/V422R/N434Y/Y436V F1066 1.45E−07 M252Y/V422S/N434Y/Y436VF1067 1.26E−07 M252Y/S424E/N434Y/Y436V F1068 1.69E−07M252Y/S424R/N434Y/Y436V F1069 1.39E−07 M252Y/N434Y/Y436V/Q438E F10701.73E−07 M252Y/N434Y/Y436V/Q438R F1071 1.24E−07 M252Y/N434Y/Y436V/Q438SF1072 1.35E−07 M252Y/N434Y/Y436V/S440E F1073 1.34E−07M252Y/N434Y/Y436V/S440R F1074 1.32E−07 S239D/M252Y/N434Y/Y436V F10751.40E−07 M252Y/K326D/L328Y/N434Y/Y436V F1076 1.27E−07S239D/M252Y/K326D/L328Y/N434Y/Y436V F1077 2.03E−06 K248N/M252Y/N434YF1078 4.70E−07 M252Y/E380N/E382S/N434Y F1079 3.44E−07M252Y/E382N/N384S/N434Y F1080 3.19E−07 M252Y/S424N/N434Y F1081 6.20E−07M252Y/N434Y/Y436N/Q438T F1082 2.76E−07 M252Y/N434Y/Q438N F1083 3.45E−07M252Y/N434Y/S440N F1094 2.60E−07 M252Y/N434Y/S442N F1095 2.86E−07M252Y/S383N/G385S/N434Y F1096 2.72E−07 M252Y/Q386T/N434Y F1097 2.82E−07M252Y/G385N/P387S/N434Y F1098 2.58E−07 S239D/M252Y/N434Y F1099 2.57E−07M252Y/K326D/L328Y/N434Y F1100 2.41E−07 S239D/M252Y/K326D/L328Y/N434YF1101 6.59E−08 S239D/M252Y/T307Q/Q311A/N434Y F1102 6.46E−08M252Y/T307Q/Q311A/K326D/L328Y/N434Y F1103 6.11E−08S239D/M252Y/T307Q/Q311A/K326D/L328Y/N434Y F1104 1.77E−07M252Y/V422E/S424R/N434Y/Y436V F1105 1.54E−07M252Y/V422S/S424R/N434Y/Y436V F1106 1.42E−07M252Y/N434Y/Y436V/Q438R/S440E F1107 1.23E−07 M252Y/V422D/N434Y/Y436VF1108 1.26E−07 M252Y/V422K/N434Y/Y436V F1109 1.27E−07M252Y/V422T/N434Y/Y436V F1110 1.33E−07 M252Y/V422Q/N434Y/Y436V

TABLE 5-27 F1111 1.65E−07 M252Y/S424K/N434Y/Y436V F1112 1.23E−07M252Y/N434Y/Y436V/Q438K F1113 1.18E−07 M252Y/N434Y/Y436V/S440D F11141.31E−07 M252Y/N434Y/Y436V/S440Q F1115 1.35E−07 M252Y/S424N/N434Y/Y436VF1116 7.44E−08 M252Y/T307Q/Q311A/S424N/N434Y F1117 4.87E−08T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V F1118 1.32E−08T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V F1119 1.03E−08T250V/M252Y/T307Q/V308P/Q311A/V422E/N434Y/Y436V F1120 1.04E−08T250V/M252Y/T307Q/V308P/Q311A/S424R/N434Y/Y436V F1121 1.04E−08T250V/M252Y/T307Q/V308P/Q311A/V422E/S424R/N434Y/Y436V F1122 1.37E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R F1123 9.55E−09T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/S440E F1124 1.22E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S440E F1125 5.18E−08M252Y/T307Q/N434Y/Y436V F1126 8.95E−08 M252Y/T307A/N434Y/Y436V F11277.94E−08 M252Y/Q311A/N434Y/Y436V F1128 1.17E−07 M252Y/Q311H/N434Y/Y436VF1129 4.48E−08 M252Y/T307Q/Q311H/N434Y/Y436V F1130 5.54E−08M252Y/T307A/Q311A/N434Y/Y436V F1131 1.29E−07L235R/S239K/M252Y/V422E/N434Y/Y436V F1132 1.40E−07L235R/S239K/M252Y/V422S/N434Y/Y436V F1133 1.58E−07L235R/S239K/M252Y/S424R/N434Y/Y436V F1134 1.66E−07L235R/S239K/M252Y/N434Y/Y436V/Q438R F1135 1.26E−07L235R/S239K/M252Y/N434Y/Y436V/S440E F1136 1.63E−07L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V F1137 1.58E−07L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V F1138 1.65E−07L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440E F1139 1.52E−07L235R/S239K/M252Y/S424N/N434Y/Y436V F1140 1.62E−07M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E F1141 1.77E−07M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E F1142 1.87E−07L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E F1143 1.98E−07L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E F1144 1.44E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/ S440E F11455.23E−08 T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F1146 6.24E−08L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F11477.19E−08 M252Y/T307Q/Q311A/N434Y/Q438R/S440E F1148 7.63E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Q438R/S440E F1151 2.51E−07L235R/S239K/M252Y/S424N/N434Y F1152 7.38E−08L235R/S239K/M252Y/T307Q/Q311A/S424N/N434Y F1153 4.85E−08L235R/S239K/T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V F1154 1.34E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436

TABLE 5-28 V F1157 2.09E−07 M252Y/N434Y/Q438R/S440E F1158 2.44E−07L235R/S239K/M252Y/N434Y/Q438R/S440E F1159 4.79E−07 S424N/N434W F11602.88E−07 V308F/S424N/N434Y F1161 1.07E−06 I332V/S424N/N434Y F11623.43E−07 P238D/T250Y/M252Y/N434Y/Y436V F1163 1.54E−07P238D/T250Y/M252Y/T307Q/Q311A/N434Y F1164 6.96E−08P238D/T250Y/M252Y/T307Q/Q311A/N434Y/Y436V F1165 1.63E−08P238D/T250Y/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1174 4.90E−07P257I/N434H F1176 1.98E−06 V308F F1178 8.72E−07 V259I/V308F/M428L F11831.28E−06 E380A/M428L/N434S F1184 1.00E−06 T307A/M428L/N434S F11859.17E−07 T307A/E380A/M428L/N434S F1188 1.72E−06 T307A/E380A/N434H F11891.57E−07 M252Y/H433D/N434Y/Y436V/Q438R/S440E F1190 2.40E−07M252Y/H433E/N434Y/Y436V/Q438R/S440E F1191 2.11E−07M252Y/N434Y/Y436V/T437A/Q438R/S440E F1192 1.27E−07M252Y/N434Y/Y436V/T437G/Q438R/S440E F1194 1.55E−07M252Y/N434Y/Y436V/Q438R/K439D/S440E F1195 1.76E−07M252Y/N434Y/Y436V/Q438R/S440E/L441A F1196 1.51E−07M252Y/N434Y/Y436V/Q438R/S440E/L441E F1197 9.46E−08M252Y/S254T/N434Y/Y436V/Q438R/S440E F1198 7.83E−08M252Y/T256E/N434Y/Y436V/Q438R/S440E F1199 6.25E−08M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1200 1.26E−07T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1201 1.07E−07T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1202 8.81E−08T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1203 1.52E−07M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1204 1.18E−07M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1205 1.98E−07T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1206 1.69E−07T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1207 1.11E−06I332E/M428L/N434S F1208 5.71E−07 L251A/M252Y/N434Y/Y436V F1211 1.23E−06L251H/M252Y/N434Y/Y436V F1213 6.33E−07 L251N/M252Y/N434Y/Y436V F12161.16E−06 L251S/M252Y/N434Y/Y436V F1217 1.14E−06 L251T/M252Y/N434Y/Y436VF1218 2.51E−07 L251V/M252Y/N434Y/Y436V F1229 2.81E−06M252Y/I253V/N434Y/Y436V F1230 1.12E−07 M252Y/N434Y/Y436V/Q438R/S440DF1231 9.73E−08 M252Y/N434Y/Y436V/Q438K/S440E

TABLE 5-29 F1232 9.79E−08 M252Y/N434Y/Y436V/Q438K/S440D F1243 1.25E−07L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1244 1.02E−07L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1245 8.20E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1246 1.73E−07L235R/S239K/T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1247 1.45E−07L235R/S239K/T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1248 1.20E−07L235R/S239K/T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F12492.06E−07 L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1250 1.66E−07L235R/S239K/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1251 2.77E−07L235R/S239K/T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1252 2.33E−07L235R/S239K/T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F12531.12E−07 L235R/S239K/M252Y/T307A/N434Y/Y436V/Q438R/S440E F1254 6.42E−08L235R/S239K/M252Y/T307Q/N434Y/Y436V/Q438R/S440E F1255 1.11E−07L235R/S239K/M252Y/Q311A/N434Y/Y436V/Q438R/S440E F1256 1.56E−07L235R/S239K/M252Y/Q311H/N434Y/Y436V/Q438R/S440E F1257 7.81E−08L235R/S239K/M252Y/T307A/Q311A/N434Y/Y436V/Q438R/S440E F1258 1.05E−07L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V/Q438R/S440E F1259 4.46E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F1260 6.53E−08L235R/S239K/M252Y/T307Q/Q311H/N434Y/Y436V/Q438R/S440E F1261 1.35E−07L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440D F1262 1.26E−07L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440E F1263 1.24E−07L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440D F1264 1.27E−07L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438R/S440E F1265 1.57E−07L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438R/S440E F1266 9.99E−08L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438R/S440E F1267 1.50E−07L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438R/S440E F1268 2.00E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440E F1269 1.69E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440D F1270 1.18E−07L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438K/S440D F1271 2.05E−07L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438R/S440E F1272 1.71E−07L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438K/S440D F1273 1.53E−07L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438K/S440D F1274 2.48E−07L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438R/S440E F1275 2.09E−07L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438K/S440D F1276 1.02E−07L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438K/S440D F1277 1.69E−07L235K/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438R/S440E F1278 1.40E−07L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438K/S440D F1279 1.23E−07L235R/S239K/M252Y/T256G/N436Y/Y436V/Q438K/S440D F1280 2.09E−07L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438R/S440E F1281 1.74E−07L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438K/S440D F1282 7.69E−08L235R/S239K/M252Y/T256N/N434V/Y436V/Q438K/S440D F1283 1.34E−07L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438R/S440E F1284 1.12E−07L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438K/S440D F1285 9.36E−08L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D

TABLE 5-30 F1286 1.57E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440E F1287 1.50E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440D F1288 7.95E−08L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D F1289 1.33E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438R/S440E F1290 1.11E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440D F1291 1.51E−07L235R/S239K/M252Y/H433D/N434Y/Y436V F1292 4.24E−07L235R/S239K/H433D/N434W/Y436V/Q438R/S440E F1293 1.61E−07L235R/S239K/M252Y/T256E/N434Y/Q438R/S440E F1294 2.00E−07L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438R/S440E F1295 9.84E−08L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438R/S440E F1296 2.27E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Q438R/S440E F1297  2.5E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438R/S440E F1298 1.47E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438R/S440E F1299 1.50E−07L235R/S239K/M252Y/T256E/N434Y/Q438K/S440D F1300 1.63E−07L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440D F1301 8.30E−08L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440D F1302 2.15E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Q438K/S440D F1303 2.10E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440D F1304 1.24E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440D F1305 2.05E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440D F1306 1.92E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440E F1307 1.44E−07L235R/S239K/M252Y/V422A/S424A/N434Y/Y436V F1308 2.06E−07L235R/S239K/M252Y/V422L/S424L/N434Y/Y436V F1309 1.26E−07L235R/S239K/M252Y/N434Y/Y436V/Q438A/S440A F1310 2.28E−07L235R/S239K/M252Y/N434Y/Y436V/Q438L/S440L F1311 1.69E−07L235R/S239K/M252Y/V422A/S424A/H433D/N434Y/Y436V F1312 1.79E−07L235R/S239K/M252Y/V422L/S424L/H433D/N434Y/Y436V F1313 1.77E−07K235R/S239K/M252Y/H433D/N434Y/Y436V/Q438A/S440A F1314 2.27E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438L/S440L F1315 1.52E−07G237K/S239K/M252Y/N434Y/Y436V F1316 1.49E−07G237R/S239K/M252Y/N434Y/Y436V F1317 1.38E−07S239K/M252Y/P329K/N434Y/Y436V F1318 1.43E−07S239K/M252Y/P329R/N434Y/Y436V F1319 2.67E−07 M252Y/L328Y/N434Y F13201.22E−07 L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440D F1321 1.03E−07L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E F1322 1.60E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440D F1323 1.49E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440E F1324 1.32E−07L234A/L235A/M252Y/N434Y/Y436V F1325 2.13E−07L234A/L235A/M252Y/N297A/N434Y/Y436V F1326 1.09E−08L234A/L235A/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1327 1.41E−08L234A/L235A/T250V/M252Y/N297A/T307Q/V308P/Q311A/N434Y/Y436V F13281.52E−07 L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438R/S440E F1329 1.29E−07L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E

TABLE 5-31 F1330 1.03E−07L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1331 7.75E−08L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F13331.23E−07 L235R/G236R/S239K/M252Y/N434Y/Y436V F1334 1.04E−07L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440D F1335 8.78E−08L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D F1336 7.18E−08L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D F1337 7.41E−08L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E F1338 1.04E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440E F1339 2.51E−07L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438K/S440E F13405.58E−08 L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E F13413.22E−07 L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438K/S440E F1342 2.51E−07L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440E F1343 2.01E−07L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438K/S440E F1344 3.96E−07L235R/S239K/M252Y/N434Y/Y436T/Q438K/S440E F1345 1.05E−07L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440E F1346 8.59E−08L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E F1347 7.14E−08L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E F1348 5.52E−08L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E F13493.36E−07 L235R/S239K/M252Y/N434Y/Y436T/Q438R/S440E F1350 1.18E−07L235R/S239K/M252Y/N434Y/Y436F/Q438K/S440E F1351 1.62E−07L235R/S239K/M252Y/N434Y/Y436F/Q438R/S440E F1352 3.93E−07L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438K/S440E F1353 1.33E−07L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438R/S440E F1354 2.29E−07L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438K/S440E F1355 2.47E−07L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438R/S440E F1356 1.58E−07G236R/M252Y/L328R/N434Y/Y436V F1357 2.81E−07L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438R/S440E F1358 9.07E−08L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438K/S440E F1359 1.28E−07L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438R/S440E F1360 3.12E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438H/S440E F1361 3.52E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438R/S440E F1362 1.41E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438K/S440E F1363 1.90E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438R/S440E F1364 7.49E−08L235R/S239K/M252Y/T256E/M434Y/Y436F/Q438K/S440E F1365 3.14E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440E F1366 1.17E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440E F1367 1.79E−07L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438R/S440E F1368 5.49E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438K/S440E F1369 7.60E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438R/S440E F1370 9.14E−08L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440E

TABLE 5-32 F1371 1.09E−07L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440E F13722.28E−07 L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438R/S440EF1373 8.67E−08L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438K/S440E F13741.20E−07 L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438R/S440EF1375 1.03E−07 L235R/S239K/M252Y/S254T/N434Y/Y436V F1376 9.09E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V F1377 8.27E−08L235R/S239K/M252Y/T256E/N434Y/Y436V F1378 3.61E−07L235R/S239K/M252Y/N434Y/Y436T F1379 2.85E−07L235R/S239K/M252Y/N434Y/Y436F F1410 1.90E−06 V308P/I332V F1411 1.70E−07V308P/I332V/M428L/N434S F1413 3.70E−08L235R/S239K/M252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V/ Q438K/S440EF1414 5.60E−08L235R/S239K/M252Y/S254T/T256E/T307Q/H433D/N434Y/Y436V/Q438K/ S440E F14155.90E−08 L235R/S239K/M252Y/S254T/T256E/Q311A/H433D/N434Y/Y436V/Q438K/S440E F1416 1.30E−08L235R/S239K/M252Y/S254T/T256E/V308P/H433D/N434Y/Y436V/Q438K/ S440E F14175.90E−08 L235R/S239K/M252Y/S254T/T256E/H433D/N434W/Y436V/Q438K/S440EF1418 7.50E−08L235R/S239K/M252Y/S254T/T256E/H433D/N434W/Y436V/Q438R/S440E F14191.50E−07 L235R/S239K/M252Y/H433D/N434W/Y436V/Q438R/S440E F1420 1.30E−07L235R/S239K/M252Y/H433D/N434W/Y436V/Q438K/S440E F1421 3.20E−08V308P/M428L/N434W F1422 1.90E−08L235R/S239K/M252Y/T256E/V308P/H433D/N434Y/Y436V/Q438R/S440E F14231.60E−08 L235R/S239K/M252Y/T256E/V302D/V308P/H433D/N434Y/Y436V/Q438R/S440E F1424 1.60E−08L235R/S239K/M252Y/T256E/V302E/V308P/H433D/N434Y/Y436V/Q438R/ S440E F14251.90E−08 L235R/S239K/M252Y/T256E/V303D/V308P/H433D/N434Y/Y436V/Q438R/S440E F1426 1.80E−08L235R/S239K/M252Y/T256E/V303E/V308P/H433D/N434Y/Y436V/Q438R/ S440E F14281.50E−08 L235R/S239K/M252Y/T256E/S304E/V308P/H433D/N434Y/Y436V/Q438R/S440E F1430 3.10E−08L235R/S239K/M252Y/T256E/V305K/V308P/H433D/N434Y/Y436V/Q438R/ S440E

TABLE 5-33 F1433 4.50E−08 L235R/S239K/M252Y/T256E/T307D/V308P/H433D/N434Y/Y436V/Q438R/S440E F1434 3.60E−08L235R/S239K/M252Y/T256E/T307E/V308P/ H433D/N434Y/Y436V/Q438R/S440E

In a non-limiting embodiment of the present invention, Fc regions inwhich at least one or more amino acids selected from the groupconsisting of amino acids at positions 257, 308, 428, and 434 accordingto EU numbering are different from the amino acids at correspondingpositions in the naturally-occurring Fc region are preferably used.Non-limiting examples of such Fc regions preferably include Fc regionscontaining at least one or more amino acids selected from the groupconsisting of:

Ala at amino acid position 257;Pro at amino acid position 308;Leu at amino acid position 428; andTyr at amino acid position 434,according to EU numbering in the Fc region.

Fcγ Receptor

Fcγ receptor refers to a receptor capable of binding to the Fc region ofmonoclonal IgG1, IgG2, IgG3, or IgG4 antibodies, and includes allmembers belonging to the family of proteins substantially encoded by anFcγ receptor gene. In human, the family includes FcγRI (CD64) includingisoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) including isoformsFcγRIIa (including allotype H131 and R131), FcγRIIb (including FcγRIIb-1and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoformFcγRIIIa (including allotype V158 and F158) and FcγRIIIb (includingallotype FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as all unidentifiedhuman FcγRs, FcγR isoforms, and allotypes thereof. However, Fcγ receptoris not limited to these examples. Without being limited thereto, FcγRincludes those derived from humans, mice, rats, rabbits, and monkeys.FcγR may be derived from any organisms. Mouse FcγR includes, withoutbeing limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), andFcγRIII-2 (FcγRIV, CD16-2), as well as all unidentified mouse FcγRs,FcγR isoforms, and allotypes thereof. Such preferred Fcγ receptorsinclude, for example, human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb(CD32), FcγRIIIa (CD16), and/or FcγRIIIb (CD16). The polynucleotidesequence and amino acid sequence of FcγRI are shown in SEQ ID NOs: 19(NM_000566.3) and 20 (NP_000557.1), respectively; the polynucleotidesequence and amino acid sequence of FcγRIIa are shown in SEQ ID NOs: 21(BC020823.1) and 27 (AAH20823.1), respectively; the polynucleotidesequence and amino acid sequence of FcγIIB are shown in SEQ ID NOs: 23(BC146678.1) and 24 (AAI46679.1), respectively; the polynucleotidesequence and amino acid sequence of FcγRIIIa are shown in SEQ ID NOs: 25(BC033678.1) and 26 (AAH33678.1), respectively; and the polynucleotidesequence and amino acid sequence of FcγRIIIb are shown in SEQ ID NOs: 27(BC128562.1) and 28 (AAI28563.1), respectively (RefSeq accession numberis shown in each parentheses). Whether an Fcγ receptor has bindingactivity to the Fc region of a monoclonal IgG1, IgG2, IgG3, or IgG4antibody can be assessed by ALPHA screen (Amplified LuminescentProximity Homogeneous Assay), surface plasmon resonance (SPR)-basedBIACORE method, and others (Proc. Natl. Acad. Sci. USA (2006) 103(11),4005-4010), in addition to the above-described FACS and ELISA formats.

Meanwhile, “Fc ligand” or “effector ligand” refers to a molecule andpreferably a polypeptide that binds to an antibody Fc region, forming anFc/Fc ligand complex. The molecule may be derived from any organisms.The binding of an Fc ligand to Fc preferably induces one or moreeffector functions. Such Fc ligands include, but are not limited to, Fcreceptors, FcγR, FcαR, FcεR, FcRn, C1q, and C3, mannan-binding lectin,mannose receptor, Staphylococcus Protein A, Staphylococcus Protein G,and viral FcγRs. The Fc ligands also include Fc receptor homologs (FcRH)(Davis et al., (2002) Immunological Reviews 190, 123-136), which are afamily of Fc receptors homologous to FcγR. The Fc ligands also includeunidentified molecules that bind to Fc.

In FcγRI (CD64) including FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16)including isoforms FcγRIIIa (including allotypes V158 and F158) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), α chainthat binds to the Fc portion of IgG is associated with common γ chainhaving ITAM responsible for transduction of intracellular activationsignal. Meanwhile, the cytoplasmic domain of FcγRII (CD32) includingisoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIccontains ITAM. These receptors are expressed on many immune cells suchas macrophages, mast cells, and antigen-presenting cells. The activationsignal transduced upon binding of these receptors to the Fc portion ofIgG results in enhancement of the phagocytic activity and inflammatorycytokine production of macrophages, mast cell degranulation, and theenhanced function of antigen-presenting cells. Fcγ receptors having theability to transduce the activation signal as described above are alsoreferred to as activating Fcγ receptors.

Meanwhile, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1and FcγRIIb-2) contains ITIM responsible for transduction of inhibitorysignals. The crosslinking between FcγRIIb and B cell receptor (BCR) on Bcells suppresses the activation signal from BCR, which results insuppression of antibody production via BCR. The crosslinking of FcγRIIIand FcγRIIb on macrophages suppresses the phagocytic activity andinflammatory cytokine production. Fcγ receptors having the ability totransduce the inhibitory signal as described above are also referred toas inhibitory Fcγ receptors.

Binding Activity to Fcγ Receptor

An embodiment of the present invention provides pharmaceuticalcompositions inducing an immune response, which comprise as an activeingredient an antigen-binding molecule containing an FcRn-binding domainthat contains an Fc region whose binding activity to human Fcγ receptorsis higher than the binding activity of the Fc of human IgG to human Fcγreceptors. Whether or not the binding activity of an Fc region to any ofthe human Fcγ receptors, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and/orFcγRIIIb, is higher than the binding activity of the Fc of human IgG tothese human Fcγ receptors can be confirmed by FACS or ELISA format asdescribed above, and also by ALPHA screen (amplified luminescentproximity homogeneous assay), BIACORE method which is based on thesurface plasmon resonance (SPR) phenomenon, or such (Proc. Natl. Acad.Sci. USA (2006) 103 (11), 4005-4010).

ALPHA screen is performed by the ALPHA technology based on the principledescribed below using two types of beads: donor and acceptor beads. Aluminescent signal is detected only when molecules linked to the donorbeads interact biologically with molecules linked to the acceptor beadsand when the two beads are located in close proximity. Excited by laserbeam, the photosensitizer in a donor bead converts oxygen around thebead into excited singlet oxygen. When the singlet oxygen diffusesaround the donor beads and reaches the acceptor beads located in closeproximity, a chemiluminescent reaction within the acceptor beads isinduced. This reaction ultimately results in light emission. Ifmolecules linked to the donor beads do not interact with moleculeslinked to the acceptor beads, the singlet oxygen produced by donor beadsdo not reach the acceptor beads and chemiluminescent reaction does notoccur.

For example, a biotin-labeled antigen-binding molecule comprising Fcregion is immobilized to the donor beads and glutathione S-transferase(GST)-tagged Fcγ receptor is immobilized to the acceptor beads. In theabsence of a competitive antigen-binding molecule comprising an alteredFc region, Fcγ receptor interacts with a polypeptide complex comprisinga wild-type Fc region, inducing a signal of 520 to 620 nm as a result. Anon-tagged antigen-binding molecule having the altered Fc regioncompetes with the antigen-binding molecule comprising a wild-type Fcregion for the interaction with Fcγ receptor. The relative bindingaffinity can be determined by quantifying the reduction of fluorescenceas a result of competition. Methods for biotinylating theantigen-binding molecules such as antibodies using Sulfo-NHS-biotin orthe like are known. Appropriate methods for adding the GST tag to an Fcγreceptor include methods that involve fusing polypeptides encoding Fcγand GST in-frame, expressing the fused gene using cells introduced witha vector to which the gene is operably linked, and then purifying usinga glutathione column. The induced signal can be preferably analyzed, forexample, by fitting to a one-site competition model based on nonlinearregression analysis using software such as GRAPHPAD PRISM (GraphPad; SanDiego).

One of the substances for observing their interaction is immobilized asa ligand onto the gold thin layer of a sensor chip. When light is shedon the rear surface of the sensor chip so that total reflection occursat the interface between the gold thin layer and glass, the intensity ofreflected light is partially reduced at a certain site (SPR signal). Theother substance for observing their interaction is injected as ananalyte onto the surface of the sensor chip. The mass of immobilizedligand molecule increases when the analyte binds to the ligand. Thisalters the refraction index of solvent on the surface of the sensorchip. The change in refraction index causes a positional shift of SPRsignal (conversely, the dissociation shifts the signal back to theoriginal position). In the Biacore system, the amount of shift describedabove (i.e., the change of mass on the sensor chip surface) is plottedon the vertical axis, and thus the change of mass over time is shown asmeasured data (sensorgram). Kinetic parameters (association rateconstant (ka) and dissociation rate constant (kd)) are determined fromthe curve of sensorgram, and affinity (KD) is determined from the ratiobetween these two constants. Inhibition assay is preferably used in theBIACORE methods. Examples of such inhibition assay are described inProc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

For the pH conditions to measure the binding activity of the Fc regionand the Fcγ receptor contained in the antigen-binding molecule of thepresent invention, conditions in an acidic pH range or in a neutral pHrange may be suitably used. The neutral pH range, as a condition tomeasure the binding activity of the Fc region and the Fcγ receptorcontained in the antigen-binding molecule of the present invention,generally indicates pH 6.7 to pH 10.0. Preferably, it is a rangeindicated with arbitrary pH values between pH 7.0 and pH 8.0; andpreferably, it is selected from pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4,pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, and pH 8.0; and particularlypreferably, it is pH 7.4, which is close to the pH of plasma (blood) invivo. Herein, the acidic pH range, as a condition for having a bindingactivity of the Fc region and the Fcγ receptor contained in theantigen-binding molecule of the present invention, generally indicatespH 4.0 to pH 6.5. Preferably, it indicates pH 5.5 to pH 6.5, andparticularly preferably, it indicates pH 5.8 to pH 6.0, which is closeto the pH in the early endosome in vivo. With regard to the temperatureused as measurement condition, the binding affinity between the Fcregion and the human Fcγ receptor can be evaluated at any temperaturebetween 10° C. and 50° C. Preferably, a temperature between 15° C. and40° C. is used to determine the binding affinity between the human Fcregion and the Fcγ receptor. More preferably, any temperature between20° C. and 35° C., such as any from 20° C., 21° C., 22° C., 23° C., 24°C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33°C., 34° C., or 35° C., can similarly be used to determine the bindingaffinity between the Fc region and the Fcγ receptor. A temperature of25° C. is a non-limiting example in an embodiment of the presentinvention.

Herein, “the binding activity of an Fc region to any of the human Fcγreceptors, FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, and/or FcγRIIIb, is higherthan the binding activity of the Fc region of human IgG to these humanFcγ receptors” means, for example, that based on the above-mentionedanalysis methods, the binding activity of an antigen-binding moleculecontaining a test Fc region is 105% or greater, preferably 110% orgreater, 120% or greater, 130% or greater, 140% or greater, particularlypreferably 150% or greater, 160% or greater, 170% or greater, 180% orgreater, 190% or greater, 200% or greater, 250% or greater, 300% orgreater, 350% or greater, 400% or greater, 450% or greater, 500% orgreater, 750% or greater, 10 times or greater, 20 times or greater, 30times or greater, 40 times or greater, and 50 times or greater relativeto the binding activity of an antigen-binding molecule containing the Fcregion of human IgG as a control.

An Fc region with higher Fcγ receptor-binding activity than the Fcγreceptor-binding activity of a naturally-occurring Fc region may beproduced by altering amino acids of the naturally-occurring Fc region.The naturally-occurring Fc region mentioned herein refers to anaturally-occurring Fc region in which the sugar chain at position 297(EU numbering) is a fucose-attached complex-type sugar chain. Whetherthe Fcγ receptor-binding activity of an Fc region is higher than that ofthe naturally-occurring Fc region may be determined appropriately byusing methods described in the “Binding activity” section mentionedabove.

In the present invention, “alteration of amino acids” or “amino acidalterations” of an Fc region include alteration to an amino acidsequence that is different from the amino acid sequence of the startingFc region. Any Fc region may be used as a starting domain as long as themodified variant of the starting Fc region can bind to human Fcγreceptors in a neutral pH range. Examples of the starting Fc regionpreferably include the Fc region of human IgG antibody, or morespecifically, a naturally-occurring Fc region in which the sugar chainat position 297 (EU numbering) is a fucose-binding complex-type sugarchain. Furthermore, an Fc region produced by further altering an alreadyaltered Fc region used as a starting Fc region may also be preferablyused as the Fc region of the present invention. The “starting Fc region”can refer to the polypeptide itself, a composition comprising thestarting Fc region, or an amino acid sequence encoding the starting Fcregion. Starting Fc regions can comprise a known IgG antibody Fc regionproduced via recombination described briefly in section “Antibodies”.The origin of starting Fc regions is not limited, and they may beobtained from human or any nonhuman organisms. Such organisms preferablyinclude mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs,goats, sheep, bovines, horses, camels and organisms selected fromnonhuman primates. In another embodiment, starting Fc regions can alsobe obtained from cynomolgus monkeys, marmosets, rhesus monkeys,chimpanzees, or humans. Starting Fc regions can be obtained preferablyfrom human IgG1; however, they are not limited to any particular IgGclass. This means that an Fc region of human IgG1, IgG2, IgG3, or IgG4can be used appropriately as a starting Fc region, and herein also meansthat an Fc region of an arbitrary IgG class or subclass derived from anyorganisms described above can be preferably used as a starting Fcregion. Examples of naturally-occurring IgG variants or modified formsare described in published documents (Curr. Opin. Biotechnol. (2009) 20(6): 685-91; Curr. Opin. Immunol. (2008) 20 (4), 460-470; Protein Eng.Des. Sel. (2010) 23 (4): 195-202; WO 2009/086320; WO 2008/092117; WO2007/041635; and WO 2006/105338); however, they are not limited to theexamples.

Examples of alterations include those with one or more mutations, forexample, mutations by substitution of different amino acid residues foramino acids of starting Fc regions, by insertion of one or more aminoacid residues into starting Fc regions, or by deletion of one or moreamino acids from starting Fc region. Preferably, the amino acidsequences of altered Fc regions comprise at least a part of the aminoacid sequence of a non-native Fc region. Such variants necessarily havesequence identity or similarity less than 100% to their starting Fcregion. In a preferred embodiment, the variants have amino acid sequenceidentity or similarity about 75% to less than 100%, more preferablyabout 80% to less than 100%, even more preferably about 85% to less than100%, still more preferably about 90% to less than 100%, and yet morepreferably about 95% to less than 100% to the amino acid sequence oftheir starting Fc region. In a non-limiting embodiment of the presentinvention, at least one amino acid is different between a modified Fcregion of the present invention and its starting Fc region. Amino aciddifference between a modified Fc region of the present invention and itsstarting Fc region can also be preferably specified based on amino aciddifferences at above-described particular amino acid positions accordingto EU numbering system.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc.Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap extension PCR canbe appropriately employed to modify the amino acids of Fc regions.Furthermore, various known methods can also be used as an amino acidmodification method for substituting amino acids by those other thannatural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35,225-249; Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). Forexample, a cell-free translation system (Clover Direct (ProteinExpress)) containing tRNAs in which amber suppressor tRNA, which iscomplementary to UAG codon (amber codon) that is a stop codon, is linkedwith an unnatural amino acid may be suitably used.

An Fc region having Fcγ receptor-binding activity in a neutral pH rangethat is contained in the antigen-binding molecules of the presentinvention may be obtained by any method, but specifically, an Fc regionhaving Fcγ receptor-binding activity in the neutral pH range may beobtained by altering amino acids of human IgG immunoglobulin used as astarting Fc region. Preferred IgG immunoglobulin Fc regions to bealtered include, for example, the Fc regions of human IgG (IgG1, IgG2,IgG3, or IgG4, and their variants). As preferred examples of the Fcregions of human IgG (IgG1, IgG2, IgG3, or IgG4, and their variants),the Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and theirvariants) may be preferably used. The structures of these Fc regions arepresented in SEQ ID NO: 11 (A is added to the N terminus of RefSeqaccession number AAC82527.1), SEQ ID NO: 12 (A is added to the Nterminus of RefSeq accession number AAB59393.1), SEQ ID NO: 13 (RefSeqaccession number CAA27268.1), and SEQ ID NO: 14 (A is added to the Nterminus of RefSeq accession number AAB59394.1). Furthermore, when usingas a test substance an antigen-binding molecule having an Fc regionproduced by altering an antibody of a certain isotype used as a startingFc region, an antigen-binding molecule having the Fc region of an IgGmonoclonal antibody of that isotype is used as a control to verifyeffects on the binding activity to Fcγ receptors by the antigen-bindingmolecule containing the altered Fc region. Antigen-binding moleculescontaining an Fc region that has been verified to have high Fcγreceptor-binding activity as described above are selected appropriately.

Amino acids at any positions may be altered to other amino acids as longas the Fc region has Fcγ receptor-binding activity in a neutral pHrange, or its Fcγ receptor-binding activity in a neutral range can beenhanced. When an antigen-binding molecule contains the Fc region ofhuman IgG1, it is preferred to include alterations that result inenhancement of Fcγ receptor-binding in a neutral pH range compared tothe binding activity of the starting Fc region of human IgG1. Amino acidalterations for enhancing Fcγ receptor-binding activity in a neutral pHrange have been reported, for example, in WO 2007/024249, WO2007/021841, WO 2006/031370, WO 2000/042072, WO 2004/029207, WO2004/099249, WO 2006/105338, WO 2007/041635, WO 2008/092117, WO2005/070963, WO 2006/020114, WO 2006/116260, and WO 2006/023403.

Examples of such amino acids that can be altered include at least one ormore amino acids selected from the group consisting of those atpositions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250,251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284,285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339,376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434,436, and 440 according to EU numbering. Alteration of these amino acidsenhances the Fcγ receptor-binding of an IgG immunoglobulin Fc region ina neutral pH range.

Particularly preferred alterations for use in the present inventioninclude the following alterations:

the amino acid at position 221 to either Lys or Tyr;the amino acid at position 222 to any one of Phe, Trp, Glu, and Tyr;the amino acid at position 223 to any one of Phe, Trp, Glu, and Lys;the amino acid at position 224 to any one of Phe, Trp, Glu, and Tyr;the amino acid at position 225 to any one of Glu, Lys, and Trp;the amino acid at position 227 to any one of Glu, Gly, Lys, and Tyr;the amino acid at position 228 to any one of Glu, Gly, Lys, and Tyr;the amino acid at position 230 to any one of Ala, Glu, Gly, and Tyr;the amino acid at position 231 to any one of Glu, Gly, Lys, Pro, andTyr;the amino acid at position 232 to any one of Glu, Gly, Lys, and Tyr;the amino acid at position 233 to any one of Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 234 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 235 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 236 to any one of Ala, Asp, Glu, Phe, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 237 to any one of Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln,

Arg, Ser, Thr, Val, Trp, and Tyr;

the amino acid at position 238 to any one of Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 239 to any one of Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr;the amino acid at position 240 to any one of Ala, Ile, Met, and Thr;the amino acid at position 241 to any one of Asp, Glu, Leu, Arg, Trp,and Tyr;the amino acid at position 243 to any one of Leu, Glu, Leu, Gln, Arg,Trp, and Tyr;the amino acid at position 244 to His;the amino acid at position 245 to Ala;the amino acid at position 246 to any one of Asp, Glu, His, and Tyr;the amino acid at position 247 to any one of Ala, Phe, Gly, His, Ile,Leu, Met, Thr, Val, and Tyr;the amino acid at position 249 to any one of Glu, His, Gln, and Tyr;the amino acid at position 250 to either Glu or Gln;the amino acid at position 251 to Phe;the amino acid at position 254 to any one of Phe, Met, and Tyr;the amino acid at position 255 to any one of Glu, Leu, and Tyr;the amino acid at position 256 to any one of Ala, Met, and Pro;the amino acid at position 258 to any one of Asp, Glu, His, Ser, andTyr;the amino acid at position 260 to any one of Asp, Glu, His, and Tyr;the amino acid at position 262 to any one of Ala, Glu, Phe, Ile, andThr;the amino acid at position 263 to any one of Ala, Ile, Met, and Thr;the amino acid at position 264 to any one of Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr;the amino acid at position 265 to any one of Ala, Leu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, andTyr;the amino acid at position 266 to any one of Ala, Ile, Met, and Thr;the amino acid at position 267 to any one of Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr;the amino acid at position 268 to any one of Asp, Glu, Phe, Gly, Ile,Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp;the amino acid at position 269 to any one of Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 270 to any one of Glu, Phe, Gly, His, Ile,Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr;the amino acid at position 271 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn,

Gln, Arg, Ser, Thr, Val, Trp, and Tyr;

the amino acid at position 272 to any one of Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 273 to either Phe or Ile;the amino acid at position 274 to any one of Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Arg,

Ser, Thr, Val, Trp, and Tyr;

the amino acid at position 275 to either Leu or Trp;the amino acid at position 276 to any one of Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 278 to any one of Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln,

Arg, Ser, Thr, Val, and Trp;

the amino acid at position 279 to Ala;the amino acid at position 280 to any one of Ala, Gly, His, Lys, Leu,Pro, Gln, Trp, and Tyr;the amino acid at position 281 to any one of Asp, Lys, Pro, and Tyr;the amino acid at position 282 to any one of Glu, Gly, Lys, Pro, andTyr;the amino acid at position 283 to any one of Ala, Gly, His, Ile, Lys,Leu, Met, Pro, Arg, and Tyr;the amino acid at position 284 to any one of Asp, Glu, Leu, Asn, Thr,and Tyr;the amino acid at position 285 to any one of Asp, Glu, Lys, Gln, Trp,and Tyr;the amino acid at position 286 to any one of Glu, Gly, Pro, and Tyr;the amino acid at position 288 to any one of Asn, Asp, Glu, and Tyr;the amino acid at position 290 to any one of Asp, Gly, His, Leu, Asn,Ser, Thr, Trp, and Tyr;the amino acid at position 291 to any one of Asp, Glu, Gly, His, Ile,Gln, and Thr;the amino acid at position 292 to any one of Ala, Asp, Glu, Pro, Thr,and Tyr;the amino acid at position 293 to any one of Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 294 to any one of Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser,

Thr, Val, Trp, and Tyr;

the amino acid at position 295 to any one of Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 296 to any one of Ala, Asp, Glu, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val;the amino acid at position 297 to any one of Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 298 to any one of Ala, Asp, Glu, Phe, His,Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr;the amino acid at position 299 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn,

Pro, Gln, Arg, Ser, Val, Trp, and Tyr;

the amino acid at position 300 to any one of Ala, Asp, Glu, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp;the amino acid at position 301 to any one of Asp, Glu, His, and Tyr;the amino acid at position 302 to Ile;the amino acid at position 303 to any one of Asp, Gly, and Tyr;the amino acid at position 304 to any one of Asp, His, Leu, Asn, andThr;the amino acid at position 305 to any one of Glu, Ile, Thr, and Tyr;the amino acid at position 311 to any one of Ala, Asp, Asn, Thr, Val,and Tyr;the amino acid at position 313 to Phe;the amino acid at position 315 to Leu;the amino acid at position 317 to either Glu or Gln;the amino acid at position 318 to any one of His, Leu, Asn, Pro, Gln,Arg, Thr, Val, and Tyr;the amino acid at position 320 to any one of Asp, Phe, Gly, His, Ile,Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 322 to any one of Ala, Asp, Phe, Gly, His,Ile, Pro, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 323 to Ile;the amino acid at position 324 to any one of Asp, Phe, Gly, His, Ile,Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr;the amino acid at position 325 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Pro,

Gln, Arg, Ser, Thr, Val, Trp, and Tyr;

the amino acid at position 326 to any one of Ala, Asp, Glu, Gly, Ile,Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 327 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr;the amino acid at position 328 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 329 to any one of Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 330 to any one of Cys, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro,

Arg, Ser, Thr, Val, Trp, and Tyr;

the amino acid at position 331 to any one of Asp, Phe, His, Ile, Leu,Met, Gln, Arg, Thr, Val, Trp, and Tyr;the amino acid at position 332 to any one of Ala, Asp, Glu, Phe, Gly,His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr;the amino acid at position 333 to any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr;the amino acid at position 334 to any one of Ala, Glu, Phe, Ile, Leu,Pro, and Thr;the amino acid at position 335 to any one of Asp, Phe, Gly, His, Ile,Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr;the amino acid at position 336 to any one of Glu, Lys, and Tyr;the amino acid at position 337 to any one of Glu, His, and Asn;the amino acid at position 339 to any one of Asp, Phe, Gly, Ile, Lys,Met, Asn, Gln, Arg, Ser, and Thr;the amino acid at position 376 to either Ala or Val;the amino acid at position 377 to either Gly or Lys;the amino acid at position 378 to Asp;the amino acid at position 379 to Asn;the amino acid at position 380 to any one of Ala, Asn, and Ser;the amino acid at position 382 to either Ala or Ile;the amino acid at position 385 to Glu;the amino acid at position 392 to Thr;the amino acid at position 396 to Leu;the amino acid at position 421 to Lys;the amino acid at position 427 to Asn;the amino acid at position 428 to either Phe or Leu;the amino acid at position 429 to Met;the amino acid at position 434 to Trp;the amino acid at position 436 to Ile; andthe amino acid at position 440 to any one of Gly, His, Ile, Leu, andTyr,according to EU numbering in the Fc region.

The number of amino acids that are altered is not particularly limited.An amino acid at one position only may be altered, or amino acids at twoor more positions may be altered. Examples of combinations of amino acidalterations at two or more positions include the combinations shown inTable 6 (Tables 6-1 to 6-3).

TABLE 6-1 Combination of amino acids Combination of amino acidsK370E/P396L/D270E S239Q/I332Q Q419H/P396L/D270E S267D/I332EV240A/P396L/D270E S267E/I332E R255L/P396L/D270E S267L/A327SR255L/P396L/D270E S267Q/A327S R255L/P396L/D270E/R292G S298A/I332ER255L/P396L/D270E S304T/I332E R255L/P396L/D270E/Y300L S324G/I332DF243L/D270E/K392N/P396L S324G/I332E F243L/R255L/D270E/P396L S324I/I332DF243L/R292P/Y300L/V305I/P396L S324I/I332E F243L/R292P/Y300L/P396LT260H/I332E F243L/R292P/Y300L T335D/I332E F243L/R292P/P396L V240I/V266IF243L/R292P/V305I V264I/I332E F243L/R292P D265F/N297E/I332ES298A/E333A/K334A D265Y/N297D/I332E E380A/T307A F243L/V262I/V264WK326M/E333S N297D/A330Y/I332E K326A/E333A N297D/T299E/I332E S317A/K353AN297D/T299F/I332E A327D/I332E N297D/T299H/I332E A330L/I332EN297D/T299I/I332E A330Y/I332E N297D/T299L/I332E E258H/I332EN297D/T299V/I332E E272H/I332E P230A/E233D/I332E E272I/N276DP244H/P245A/P247V E272R/I332E S239D/A330L/I332E E283H/I332ES239D/A330Y/I332E E293R/I332E S239D/H268E/A330Y F241L/V262IS239D/I332E/A327A F241W/F243W S239D/I332E/A330I

TABLE 6-2 F243L/V264I S239D/N297D/I332E H268D/A330Y S239D/S298A/I332EH268E/A330Y S239D/V264I/I332E K246H/I332E S239E/N297D/I332E L234D/I332ES239E/V264I/I332E L234E/I332E S239N/A330L/I332E L234G/I332ES239N/A330Y/I332E L234I/I332E S239N/S298A/I332E L234I/L235DS239Q/V264I/I332E L234Y/I332E V264E/N297D/I332E L235D/I332EV264I/A330L/I332E L235E/I332E V264I/A330Y/I332E L235I/I332EV264I/S298A/I332E L235S/I332E Y296D/N297D/I332E L328A/I332DY296E/N297D/I332E L328D/I332D Y296H/N297D/I332E L328D/I332EY296N/N297D/I332E L328E/I332D Y296Q/N297D/I332E L328E/I332EY296T/N297D/I332E L328F/I332D D265Y/N297D/T299L/I332E L328F/I332EF241E/F243Q/V262T/V264E L328H/I332E F241E/F243R/V262E/V264R L328I/I332DF241E/F243Y/V262T/V264R L328I/I332E F241L/F243L/V262I/V264I L328M/I332DF241R/F243Q/V262T/V264R L328M/I332E F241S/F243H/V262T/V264T L328N/I332DF241W/F243W/V262A/V264A L328N/I332E F241Y/F243Y/V262T/V264T L328Q/I332DI332E/A330Y/H268E/A327A L328Q/I332E N297D/I332E/S239D/A330L L328T/I332DN297D/S298A/A330Y/I332E L328T/I332E S239D/A330Y/I332E/K326E L328V/I332DS239D/A330Y/I332E/K326T L328V/I332E S239D/A330Y/I332E/L234I L328Y/I332DS239D/A330Y/I332E/L235D

TABLE 6-3 L328Y/I332E S239D/A330Y/I332E/V240I N297D/I332ES239D/A330Y/I332E/V264T N297E/I332E S239D/A330Y/I332E/V266I N297S/I332ES239D/D265F/N297D/I332E P227G/I332E S239D/D265H/N297D/I332E P230A/E233DS239D/D265I/N297D/I332E Q295E/I332E S239D/D265L/N297D/I332E R255Y/I332ES239D/D265T/N297D/I332E S239D/I332D S239D/D265V/N297D/I332E S239D/I332ES239D/D265Y/N297D/I332E S239D/I332N S239D/I332E/A330Y/A327A S239D/I332QS239D/I332E/H268E/A327A S239E/D265G S239D/I332E/H268E/A330Y S239E/D265NS239D/N297D/I332E/A330Y S239E/D265Q S239D/N297D/I332E/K326E S239E/I332DS239D/N297D/I332E/L235D S239E/I332E S239D/V264I/A330L/I332E S239E/I332NS239D/V264I/S298A/I332E S239E/I332Q S239E/V264I/A330Y/I332E S239N/I332DF241E/F243Q/V262T/V264E/I332E S239N/I332E F241E/F243R/V262E/V264R/I332ES239N/I332N F241E/F243Y/V262T/V264R/I332E S239N/I332QF241R/F243Q/V262T/V264R/I332E S239Q/I332D S239D/I332E/H268E/A330Y/A327AS239Q/I332E S239E/V264I/S298A/A330Y/I332E S239Q/I332NF241Y/F243Y/V262T/V264T/N297D/I332E S267E/L328F G236D/S267E S239D/S267EFc Region with Modified Sugar Chains

Fc regions provided by the present invention may include Fc regions thatare modified so that the percentage of Fc regions to which afucose-deficient sugar-chain is attached will become higher, or that thepercentage of Fc regions to which bisecting N-acetylglucosamine is addedwill become higher. It is known that the affinity of an antibody Fcregion for FcγRIIIa is enhanced when the fucose residue is removed fromN-acetylglucosamine at the reducing end of an N-glycoside-linkedcomplex-type sugar chain bound to the antibody Fc region (Non-PatentDocument 20). IgG1 antibodies containing such Fc regions are known tohave enhanced ADCC activity, which will be described later; therefore,antigen-binding molecules containing such Fc regions are also useful asantigen-binding molecules to be included in the pharmaceuticalcompositions of the present invention. Known examples of antibodies inwhich the fucose residue has been removed from N-acetylglucosamine atthe reducing end of an N-glycoside-linked complex-type sugar chain boundto the antibody Fc region are the following: glycosylation-modifiedantibodies (for example, WO 1999/054342);

antibodies lacking fucose attached to sugar chains (for example, WO2000/061739, WO 2002/031140, and WO 2006/067913);antibodies having a sugar chain with bisecting GlcNAc (for example, WO2002/079255), and such. Methods for producing these antibodies may alsobe applied to methods for producing antigen-binding molecules containingan altered Fc region which has been modified so that the percentage ofthe Fc region to which a fucose-deficient sugar-chain is attached willbecome higher, or that the percentage of the Fc region to whichbisecting N-acetylglucosamine is added will become higher.

Antigen-Binding Molecules

In the present invention, this term is used in the broadest sense torefer to molecules containing an antigen-binding domain whoseantigen-binding activity changes depending on ion concentrationconditions, and an FcRn-binding domain having FcRn-binding activity in aneutral pH range. Specifically, various molecular types may be includedas long as they show antigen-binding activity. Molecules in which anantigen-binding domain is linked to an Fc region include, for example,antibodies. Antibodies may include single monoclonal antibodies(including agonistic antibodies and antagonistic antibodies), humanantibodies, humanized antibodies, chimeric antibodies, and such.Alternatively, when used as antibody fragments, they preferably includeantigen-binding domains and antigen-binding fragments (for example, Fab,F(ab′)2, scFv, and Fv). Scaffold molecules where three dimensionalstructures, such as already-known stable α/β barrel protein structure,are used as a scaffold (base) and only some portions of the structuresare made into libraries to construct antigen-binding domains are alsoincluded in antigen-binding molecules of the present invention.

An antigen-binding molecule of the present invention may contain atleast some portions of an Fc region that mediates the binding to FcRnand Fcγ receptor. In a non-limiting embodiment, the antigen-bindingmolecule includes, for example, antibodies and Fc fusion proteins. Afusion protein refers to a chimeric polypeptide comprising a polypeptidehaving a first amino acid sequence that is linked to a polypeptidehaving a second amino acid sequence that would not naturally link innature. For example, a fusion protein may comprise the amino acidsequence of at least a portion of an Fc region (for example, a portionof an Fc region responsible for the binding to FcRn or a portion of anFc region responsible for the binding to Fcγ receptor) and anon-immunoglobulin polypeptide containing, for example, the amino acidsequence of the ligand-binding domain of a receptor or areceptor-binding domain of a ligand. The amino acid sequences may bepresent in separate proteins that are transported together to a fusionprotein, or generally may be present in a single protein; however, theyare included in a new rearrangement in a fusion polypeptide. Fusionproteins can be produced, for example, by chemical synthesis, or bygenetic recombination techniques to express a polynucleotide encodingpeptide regions in a desired arrangement.

Respective domains of the present invention can be linked together vialinkers or directly via polypeptide binding. The linkers comprisearbitrary peptide linkers that can be introduced by genetic engineering,synthetic linkers, and linkers disclosed in, for example, ProteinEngineering (1996) 9(3), 299-305. However, peptide linkers are preferredin the present invention. The length of the peptide linkers is notparticularly limited, and can be suitably selected by those skilled inthe art according to the purpose. The length is preferably five aminoacids or more (without particular limitation, the upper limit isgenerally 30 amino acids or less, preferably 20 amino acids or less),and particularly preferably 15 amino acids.

For example, such peptide linkers preferably include:

Ser Gly⋅Ser Gly⋅Gly⋅Ser Ser⋅Gly⋅Gly Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 29)Ser⋅Gly⋅Gly⋅Gly (SEQ ID NO: 30) Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 31)Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 32) Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO:33) Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 34) Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser(SEQ ID NO: 35) Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 36)(Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 31))n (Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO:32))n

where n is an integer of 1 or larger. The length or sequences of peptidelinkers can be selected accordingly by those skilled in the artdepending on the purpose.

Synthetic linkers (chemical crosslinking agents) is routinely used tocrosslink peptides, and for example:

N-hydroxy succinimide (NHS),disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidyl propionate) (DSP),dithiobis(sulfosuccinimidyl propionate) (DTSSP),ethylene glycol bis(succinimidyl succinate) (EGS),ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES),and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).These crosslinking agents are commercially available.

When multiple linkers for linking the respective domains are used, theymay all be of the same type, or may be of different types.

In addition to the linkers exemplified above, linkers with peptide tagssuch as His tag, HA tag, myc tag, and FLAG tag may also be suitablyused. Furthermore, hydrogen bonding, disulfide bonding, covalentbonding, ionic interaction, and properties of binding with each other asa result of combination thereof may be suitably used. For example, theaffinity between CH1 and CL of antibody may be used, and Fc regionsoriginating from the above-described bispecific antibodies may also beused for hetero Fc region association. Moreover, disulfide bonds formedbetween domains may also be suitably used.

In order to link respective domains via peptide linkage, polynucleotidesencoding the domains are linked together in frame. Known methods forlinking polynucleotides in frame include techniques such as ligation ofrestriction fragments, fusion PCR, and overlapping PCR. Such methods canbe appropriately used alone or in combination to constructantigen-binding molecules of the present invention. In the presentinvention, the terms “linked” and “fused”, or “linkage” and “fusion” areused interchangeably. These terms mean that two or more elements orcomponents such as polypeptides are linked together to form a singlestructure by any means including the above-described chemical linkingmeans and genetic recombination techniques. Fusing in frame means, whentwo or more elements or components are polypeptides, linking two or moreunits of reading frames to form a continuous longer reading frame whilemaintaining the correct reading frames of the polypeptides. When twomolecules of Fab are used as an antigen-binding domain, an antibody,which is an antigen-binding molecule of the present invention where theantigen-binding domain is linked in frame to an Fc region via peptidebond without linker, can be used as a preferred antigen-binding moleculeof the present invention.

Neutralizing Activity

An embodiment of the present invention provides pharmaceuticalcompositions that induce an immune response, which comprise as an activeingredient an antigen-binding molecule having neutralizing activityagainst an antigen, and containing an antigen-binding domain whoseantigen-binding activity changes depending on ion concentrationconditions and an FcRn-binding domain having FcRn-binding activity in aneutral pH range. Generally, neutralizing activity refers to activity ofinhibiting the biological activity of a ligand, such as viruses andtoxins, having biological activity on cells. Thus, substances havingneutralizing activity refer to substances that bind to the ligand or thereceptor to which the ligand binds, and inhibits the binding between theligand and the receptor. Receptors blocked from binding with the ligandby the neutralizing activity will not be able to exhibit biologicalactivity through this receptor. When the antigen-binding molecule is anantibody, such an antibody having neutralizing activity is generallycalled a neutralizing antibody. Neutralizing activity of a testsubstance may be measured by comparing the biological activity in thepresence of a ligand between when the test substance is present andabsent.

For example, major possible ligands for the IL-6 receptor preferablyinclude IL-6 as shown in SEQ ID NO: 37. The IL-6 receptor, which is anI-type membrane protein with its amino terminus forming theextracellular domain, forms a hetero-tetramer with a gp130 receptorwhich has been induced to dimerize by IL-6 (Heinrich et al. (Biochem. J.(1998) 334, 297-314)). Formation of the heterotetramer activates Jakwhich is associated with the gp130 receptor. Jak undergoesautophosphorylation and phosphorylates the receptor. The phosphorylationsite of the receptor and Jak serves as a binding site for SH2-carryingmolecules belonging to the Stat family such as Stat3; MAP kinase;PI3/Akt; and other SH2-carrying proteins and adapters. Next, Stat boundto the gp130 receptor is phosphorylated by Jak. The phosphorylated Statdimerizes and moves into the nucleus, and regulates the transcription oftarget genes. Jak or Stat can also be involved in signal cascades viareceptors of other classes. Deregulated IL-6 signal cascades areobserved in inflammation and pathological conditions of autoimmunediseases, and cancers such as prostate cancer and multiple myeloma.Stat3 which may act as an oncogene is constitutively activated in manycancers. In prostate cancer and multiple myeloma, there is a crosstalkbetween the signaling cascade via the IL-6 receptor and the signalingcascade via the epithelial growth factor receptor (EGFR) family members(Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2), 55-66)).

Such intracellular signaling cascades are different for each cell type;therefore, appropriate target molecules can be determined for eachtarget cell of interest, and are not limited to the above-mentionedfactors. Neutralization activity can be evaluated by measuring theactivation of in vivo signaling. Furthermore, the activation of in vivosignaling can be detected by using as an index the action of inducingthe transcription of a target gene that exists downstream of the in vivosignaling cascade. Change in the transcription activity of the targetgene can be detected by the principle of reporter assays. Specifically,a reporter gene such as green fluorescence protein (GFP) or luciferaseis placed downstream of a promoter region or a transcription factor ofthe target gene, its reporter activity is measured, and thereby changein the transcription activity can be measured as the reporter activity.Commercially available kits for measuring the activation of in vivosignaling can be used appropriately (for example, Mercury PathwayProfiling Luciferase System (Clontech)).

Furthermore, for methods of measuring the activity of neutralizingreceptors/ligands of the EGF receptor family and such, which normallyact on signaling cascades that work toward promoting cell proliferation,the neutralization activity of neutralizing antibodies can be evaluatedby measuring the proliferation activity of target cells. For example,when cells are promoted to proliferate by growth factors of the EGFfamily such as HB-EGF, the inhibitory effect on the proliferation ofsuch cells based on the neutralizing activity of an anti-HB-EGF antibodycan be suitably evaluated or measured by the following methods: Forevaluating or measuring the cell proliferation inhibitory activity invitro, a method of measuring the incorporation of [³H]-labeled thymidineadded to the medium by viable cells as an index of DNA replicationability is used. As more convenient methods, a dye exclusion method, inwhich the ability of a cell to exclude a dye such as trypan blue fromthe cell is measured under the microscope, and the MTT method, are used.The latter method makes use of the ability of viable cells to convertMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide),which is a tetrazolium salt, to a blue formazan product. Morespecifically, a test antibody is added as well as a ligand to theculture solution of a test cell, and after a certain period of time, theMTT solution is added to the culture solution, and this is left to standfor a while for incorporation of MTT into the cell. As a result, MTT,which is a yellow compound, is converted to a blue compound by theaction of succinate dehydrogenase in the mitochondria of the cell. Afterdissolving this blue product for coloration, its absorbance is measuredand used as an index for the number of viable cells. In addition to MTT,reagents such as MTS, XTT, WST-1, and WST-8 are also commerciallyavailable (Nacalai Tesque, and such) and can be suitably used. Formeasuring the activity, a binding antibody which is of the same isotypeas the anti-HB-EGF antibody but does not have the cell proliferationinhibitory activity can be used as a control antibody in the same manneras the anti-HB-EGF antibody, and the activity can be determined when theanti-HB-EGF antibody shows stronger cell proliferation inhibitoryactivity than the control antibody.

Cells that can be preferably used for evaluating the activity include,for example, cells promoted to proliferate by HB-EGF such as ovariancancer cell line RMG-1, and mouse Ba/F3 cells which have beentransformed by a vector for expressing a gene encoding hEGFR/mG-CSFR,which is a fusion protein in which the extracellular domain of humanEGFR is fused in frame with the intracellular domain of the mouse GCSFreceptor. In this way, those skilled in the art can appropriately selectcells to be used for evaluating the activity and use them to measure thecell proliferation activity as mentioned above.

Cytotoxic Activity

An embodiment of the present invention provides pharmaceuticalcompositions that induce an immune response, which comprise as an activeingredient an antigen-binding molecule that has cytotoxic activityagainst cells expressing an antigen, and contains an antigen-bindingdomain whose antigen-binding activity changes depending on ionconcentration conditions and an FcRn-binding domain having FcRn-bindingactivity in a neutral pH range. In the present invention, the cytotoxicactivity includes, for example, antibody-dependent cell-mediatedcytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC)activity. In the present invention, CDC activity refers to cytotoxicactivity mediated by the complement system. ADCC activity refers to theactivity of damaging a target cell when a specific antigen-bindingmolecule attaches to the surface antigen of an antigen-expressing celland then a Fcγ receptor-expressing cell (immune cell, or such) binds tothe Fc portion of the antigen-binding molecule via the Fcγ receptor.Whether an antigen-binding molecule of interest has ADCC activity or CDCactivity can be determined using known methods (for example, CurrentProtocols in Immunology, Chapter 7. Immunologic studies in humans,Coligan et al. Ed. (1993)).

First, specifically, effector cells, complement solution, and targetcells are prepared.

(1) Preparation of Effector Cells

Spleen is removed from a CBA/N mouse or the like, and spleen cells areisolated in RPMI1640 medium (Invitrogen). After washing the cells withthe same medium containing 10% fetal bovine serum (FBS, HyClone), theconcentration of the washed spleen cells may be adjusted to 5×10⁶/mL toprepare effector cells.

(2) Preparation of Complement Solution

Baby Rabbit Complement (CEDARLANE) is diluted 10-fold in a culturemedium (Invitrogen) containing 10% FBS to prepare a complement solution.

(3) Preparation of Target Cells

Target cells can be radioactively labeled by culturing cells expressingan antigen with 0.2 mCi sodium chromate-⁵¹Cr (GE HealthcareBio-Sciences) in a DMEM medium containing 10% FBS for one hour at 37° C.After radioactive labeling, the cells are washed three times withRPMI1640 medium containing 10% FBS, and the target cells can be preparedby adjusting the cell concentration to 2×10⁵/mL.

ADCC activity or CDC activity can be measured by the method describedbelow. In the case of measuring ADCC activity, 50 μL each of the targetcell and antigen-binding molecule are added to a 96-well U-bottom plate(Becton Dickinson), and allowed to react for 15 minutes on ice.Thereafter, 100 μL of effector cells are added to the plate and left tostand in a carbon dioxide incubator for four hours. The finalconcentration of the antibody may be adjusted to, for example, 0 or 10μg/mL. After being left to stand, 100 μL of the supernatant is collectedfrom each well, and the radioactivity is measured with a gamma counter(COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument Company). Themeasured value is used to calculate cytotoxic activity (%) according tothe formula: (A−C)/(B−C)×100. A represents the radioactivity (cpm) ineach sample, B represents the radioactivity (cpm) in a sample to which1% NP-40 (Nacalai Tesque) has been added, and C represents theradioactivity (cpm) of a sample containing the target cells only.

Meanwhile, in the case of measuring CDC activity, 50 μL each of thetarget cell and antigen-binding molecule are added to a 96-wellflat-bottomed plate (Becton Dickinson), and allowed to react for 15minutes on ice. Thereafter, 100 μL of the complement solution is addedto the plate, and left to stand in a carbon dioxide incubator for fourhours. The final concentration of the antibody may be adjusted to, forexample, 0 or 3 μg/mL. After being left to stand, 100 μL of supernatantis collected from each well, and the radioactivity is measured with agamma counter. The cytotoxic activity can be calculated in the same wayas in the measurement of ADCC activity.

Immune Response

Anon-limiting embodiment of the present invention providespharmaceutical compositions that induce an immune response to anantigen, which comprise as an active ingredient an antigen-bindingmolecule containing an antigen-binding domain whose antigen-bindingactivity changes depending on ion concentration conditions and anFcRn-binding domain having FcRn-binding activity in a neutral pH range.

Whether an immune response has been induced may be evaluated bymeasuring the response in a living organism that has received apharmaceutical composition that induces an immune response against theaforementioned antigen and contains an antigen-binding molecule as anactive ingredient. Examples of the response in the living organismmainly include two immune responses: cellular immunity (induction ofcytotoxic T cells that recognize a peptide fragment of the antigen boundto MHC class I) and humoral immunity (induction of the production ofantigen-binding antibodies). Methods for evaluating induction of humoralimmunity (immune response) include methods for evaluating production ofantibodies against an antigen in vivo.

Whether humoral immunity has been induced by a pharmaceuticalcomposition of the present invention that induces an immune response maybe evaluated by administering the pharmaceutical composition to a livingorganism and, in the peripheral blood isolated from the organism,detecting antibodies raised by the organism against the antigen targetedby the antigen-binding molecule contained in the pharmaceuticalcomposition. The titer of an antibody against an antigen can be measuredby applying the methods for measuring molecules that specifically bindto an administered molecule using ELISA, ECL, SPR, which are known tothose skilled in the art (J. Pharm. Biomed. Anal. 2011 Jul. 15;55(5):878-88).

Whether cellular immunity has been acquired due to a pharmaceuticalcomposition of the present invention that induces an immune response maybe evaluated by administering the pharmaceutical composition to a livingorganism and, in the peripheral blood isolated from the organism,detecting a subset of CD8-expressing T cells which have a memory-typephenotype and are specific to the antigen targeted by theantigen-binding molecule contained in the pharmaceutical composition. Apopulation of CD8-expressing cells having a memory-type phenotype is aheterogeneous cell population. Specifically, it includes central memorycells, which rapidly divide in response to the antigen, and effectormemory cells, which show the memory of effector functions such ascytotoxicity. These subsets are not mutually exclusive. That is, thecells may divide rapidly but may also damage target cells presenting theantigen.

There is a commercially available kit (Cytokine Secretion Assay—CellEnrichment and Detection Kit (Miltenyi Biotec)) for detecting cytokinesproduced as a result of performing expansion culture of such a subset ofantigen-specific, CD8-expressing T cells having a memory-type phenotype.Protocols for isolating such an antigen-specific population are alsoprovided. By using such a kit, both antigen-specific central memorycells and effector memory

T cells can be grown efficiently in vitro. Antigen-presenting cells thatcan stimulate the proliferation of a subset of such T cells may beisolated from the peripheral blood obtained from the organism to whichthe aforementioned immune-response-inducing pharmaceutical compositionhas been administered. Dendritic cells pulsed with the antigen ordendritic cells transfected by the antigen (Overes et al. (J.Immunother. (2009) 32, 539-551) may be used as antigen-presenting cells.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticalcompositions that induce an immune response to an antigen, whichcomprise as an active ingredient an antigen-binding molecule containingan antigen-binding domain whose antigen-binding activity changesdepending on ion concentration conditions and an FcRn-binding domainhaving FcRn-binding activity in a neutral pH range. A differentembodiment of the present invention relates to cell growth inhibitors oranti-cancer agents that induce an immune response to the aforementionedantigen, which comprise the antigen-binding molecule as an activeingredient. A pharmaceutical composition, a cell growth inhibitor, or ananti-cancer agent of the present invention is preferably administered toa subject affected with infection by foreign biological species or withcancer, or a subject that may experience recurrence.

In an embodiment of the present invention, a pharmaceutical composition,cell growth inhibitor, or an anticancer agent that induces an immuneresponse to the aforementioned antigen, which comprises as an activeingredient an antigen-binding molecule containing an antigen-bindingdomain whose antigen-binding activity changes depending on ionconcentration conditions and an FcRn-binding domain having FcRn-bindingactivity in a neutral pH range, may be expressed as use of saidantigen-binding molecule in producing said pharmaceutical composition,cell growth inhibitor, or anticancer agent.

In another embodiment of the present invention, it may also be expressedas a method for inducing an immune response to an antigen, whichcomprises the step of administering a pharmaceutical composition, a cellgrowth inhibitor, or an anticancer agent which comprises as an activeingredient an antigen-binding molecule containing an antigen-bindingdomain whose antigen-binding activity changes depending on ionconcentration conditions and an FcRn-binding domain having FcRn-bindingactivity in a neutral pH range.

In another embodiment of the present invention, it may be expressed asan antigen-binding molecule which contains an antigen-binding domainwhose antigen-binding activity changes depending on ion concentrationconditions and an FcRn-binding domain having FcRn-binding activity in aneutral pH range, for use in inducing an immune response to an antigen.

In another embodiment of the present invention, it may be expressed as aprocess for producing a pharmaceutical composition, a cell growthinhibitor, or an anticancer agent that induces an immune response to anantigen, which comprises the step of using an antigen-binding moleculecontaining an antigen-binding domain whose antigen-binding activitychanges depending on ion concentration conditions and an FcRn-bindingdomain having FcRn-binding activity in a neutral pH range.

In the present invention, the phrase “comprises as an active ingredientan antigen-binding molecule containing an antigen-binding domain whoseantigen-binding activity changes depending on ion concentrationconditions and an FcRn-binding domain having FcRn-binding activity in aneutral pH range” means comprising said antigen-binding molecule as amajor active component, and does not limit the content ratio of theantigen-binding molecule.

Furthermore, the present invention may provide pharmaceuticalcompositions, cell growth inhibitors, and anticancer agents that induceimmune response to an antigen, which comprise as an active ingredient,in addition to an antigen-binding molecule that is not bound to theantigen, an antigen-binding molecule that has already bound to theantigen. Moreover, the present invention provides methods for inducingan immune response to an antigen, which comprise administering, inaddition to an antigen-binding molecule that is not bound to theantigen, an antigen-binding molecule that has already bound to theantigen.

Furthermore, pharmaceutical compositions, cell growth inhibitors, andanticancer agents of the present invention may include differentantigen-binding molecules when necessary. For example, a cocktail ofdifferent antigen-binding molecules of the present invention that bindto the same antigen may enhance the action of inducing an immuneresponse, cytotoxic activity, or neutralization activity against cellsexpressing the antigen, resulting in increased therapeutic effectsagainst diseases caused by the cells expressing the antigen.Alternatively, a pharmaceutical composition, cell growth inhibitor, oranticancer agent of the present invention which comprises anantigen-binding molecule of the present invention containing anantigen-binding domain that binds to an antigen expressed by cellscausing a disease to be treated, and also comprises an antigen-bindingmolecule of the present invention containing an antigen binding domainthat binds to another antigen expressed by the cells causing the samedisease, is administered to increase therapeutic effects on the disease.

If necessary, a pharmaceutical composition, a cell growth inhibitor, oran anticancer agent of the present invention can be encapsulated intomicrocapsules (microcapsules made of hydroxymethylcellulose, gelatin,poly[methyl methacrylate], or such), and prepared as colloidal drugdelivery systems (such as liposomes, albumin microspheres,microemulsion, nanoparticles, and nanocapsules) (see, for example,“Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980)).Methods for preparing a drug as a controlled-release drug are alsoknown, and such methods may be applied to the pharmaceuticalcompositions, cell growth inhibitors, and anticancer agents of thepresent invention (J. Biomed. Mater. Res. (1981) 15, 267-277; Chemtech.(1982) 12: 98-105; U.S. Pat. No. 3,773,919; European Patent PublicationsEP 58481 and EP 133988; Biopolymers (1983) 22, 547-556).

The pharmaceutical compositions, cell growth inhibitors, and anticanceragents of the present invention can be administered to patients eitherorally or parenterally. Parenteral administration is preferred. Suchadministration methods specifically include administration by injection,transnasal administration, pulmonary administration, and transdermaladministration. For administration by injection, a pharmaceuticalcomposition, a cell growth inhibitor, or an anticancer agent of thepresent invention can be systemically or locally administered by, forexample, intravenous injection, intramuscular injection, intraperitonealinjection, and subcutaneous injection. The method of administration canbe selected appropriately according to the age and symptoms of thepatient. The dose can be selected, for example, within the range from0.0001 mg to 1000 mg per kilogram body weight per administration.Alternatively, the dose may be selected, for example, within the rangefrom 0.001 mg/body to 100000 mg/body per patient. However, thepharmaceutical compositions, cell growth inhibitors, or anticanceragents of the present invention are not limited to these doses.

The pharmaceutical compositions, cell growth inhibitors, and anticanceragents of the present invention can be formulated according toconventional methods (for example, Remington's Pharmaceutical Science,latest edition, Mark Publishing Company, Easton, U.S.A), and may alsocontain pharmaceutically acceptable carriers and additives. Examplesinclude surfactants, excipients, coloring agents, flavoring agents,preservatives, stabilizers, buffers, suspending agents, isotonizationagents, binders, disintegrants, lubricants, fluidity-promoting agents,and corrigents. Without limitation to these, other commonly usedcarriers can be suitably used. Specific examples of carriers includelight anhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, polyvinylacetal diethylaminoacetate,polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides,polyoxyethylene hardened castor oil 60, saccharose, carboxymethylcellulose, corn starch, inorganic salts, and such.

Amino acids contained in the amino acid sequences in the presentinvention may be post-translationally modified (for example, themodification of an N-terminal glutamine into a pyroglutamic acid bypyroglutamylation is well-known to those skilled in the art). Naturally,such post-translationally modified amino acids are included in the aminoacid sequences in the present invention.

Methods for Producing Pharmaceutical Compositions

Anon-limiting embodiment of the present invention provides methods forproducing an antigen-binding molecule that induces an immune response,which comprise imparting FcRn-binding activity in a neutral pH range toan FcRn-binding domain that is contained in an antigen-binding moleculecontaining an antigen-binding domain whose antigen-binding activitychanges depending on ion concentration conditions.

In the production methods of the present invention, when theFcRn-binding activity of the FcRn-binding domain that is contained in anantigen-binding molecule containing an antigen-binding domain whoseantigen-binding activity changes depending on ion concentrationconditions is weak or not detected in a neutral pH range, FcRn-bindingactivity in the neutral pH range can be imparted to the FcRn-bindingdomain to produce an antigen-binding molecule of the present invention.

For example, when an antigen-binding domain containing the heavy andlight chain variable regions of an anti-FcRn antibody is used as an FcRnbinding domain, it is possible to obtain an FcRn-binding domain havingFcRn-binding activity in a neutral pH range according to theaforementioned method for obtaining an antigen-binding domain whoseantigen-binding activity changes depending on ion concentrationconditions. When an Fc region whose

FcRn-binding activity in a neutral pH range is weak or undetectable isused as an FcRn-binding domain, an antigen-binding molecule containingan Fc region with desired FcRn-binding activity may be obtained byaltering amino acids of the Fc region contained in the antigen-bindingmolecule. Amino acid alterations of the Fc region that result in suchdesired binding activity may be identified by comparing the FcRn-bindingactivity in a neutral pH range before and after amino acid alteration.Those skilled in the art can carry out appropriate amino acidalterations using known methods such as overlap extension PCR orsite-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA(1985) 82, 488-492)) similarly to the aforementioned methods used toalter antigen-binding activity.

An Fc region having FcRn-binding activity in a neutral pH range that iscontained in an antigen-binding molecule of the present invention may beobtained by any methods, but specifically, an FcRn-binding domain havingFcRn-binding activity or enhanced FcRn-binding activity in a neutral pHrange may be obtained by altering amino acids of human IgGimmunoglobulin used as a starting Fc region. Examples of preferred IgGimmunoglobulin Fc regions to be altered include the Fc region of humanIgG (IgG1, IgG2, IgG3, or IgG4, and their variants). Amino acids at anypositions may be altered to other amino acids as long as the Fc regionhas FcRn-binding activity in a neutral pH range or its humanFcRn-binding activity in a neutral range can be enhanced. When anantigen-binding molecule contains the Fc region of human IgG1, it ispreferred to include alterations that result in enhancement ofFcRn-binding in a neutral pH range compared to the binding activity ofthe starting Fc region of human IgG1. KD values for FcRn in a neutral pHrange are determined as mentioned above by the method described in TheJournal of Immunology (2009) 182: 7663-7671 (antigen-binding moleculesare immobilized onto a chip and human FcRn is allowed to flow as ananalyte).

Examples of preferred IgG immunoglobulin Fc regions to be alteredinclude the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and theirvariants). Amino acids at any positions may be altered to other aminoacids as long as the Fc region has FcRn-binding activity in a neutral pHrange or its human FcRn-binding activity in the neutral range can beenhanced. When an antigen-binding molecule contains the Fc region ofhuman IgG1, it is preferred to include alterations that result inenhancement of FcRn-binding in a neutral pH range compared to thebinding activity of the starting Fc region of human IgG1. Examples ofsuch a modified Fc region include altered Fc regions in which aminoacids such as those listed in Table 5 above have been altered and whichhave binding activity in a neutral pH range.

A non-limiting embodiment of the present invention provides methods forproducing a pharmaceutical composition that induces an immune response,which comprise the steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain under a high calcium ion concentration condition;

(b) determining the antigen-binding activity of the antigen-bindingdomain under a low calcium ion concentration condition;

(c) selecting the antigen-binding domain whose antigen-binding activitydetermined in (a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding an FcRn-binding domainhaving FcRn-binding activity in a neutral pH range;

(e) culturing cells into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from the culture fluid of thecells cultured in (e).

Another non-limiting embodiment of the present invention also providesmethods for producing a pharmaceutical composition that induces animmune response, which comprise the steps of:

(a) determining the antigen-binding activity of an antibody under a highcalcium ion concentration condition;

(b) determining the antigen-binding activity of the antibody under a lowcalcium ion concentration condition;

(c) selecting the antibody whose antigen-binding activity determined in(a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domain of theantibody selected in (c) to a polynucleotide encoding an FcRn-bindingdomain having FcRn-binding activity in a neutral pH range;

(e) culturing cells into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from the culture fluid of thecells cultured in (e).

Furthermore, a non-limiting embodiment of the present invention providesmethods for producing an antigen-binding molecule, which comprise thesteps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain in a neutral pH range;

(b) determining the antigen-binding activity of the antigen-bindingdomain in an acidic pH range;

(c) selecting the antigen-binding domain whose antigen-binding activitydetermined in (a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding an FcRn-binding domainhaving FcRn-binding activity in a neutral pH range;

(e) culturing cells into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from the culture fluid of thecells cultured in (e).

Furthermore, another non-limiting embodiment of the present inventionprovides methods for producing an antigen-binding molecule, whichcomprise the steps of:

(a) determining the antigen-binding activity of an antibody in a neutralpH range;

(b) determining the antigen-binding activity of the antibody in anacidic pH range;

(c) selecting the antibody whose antigen-binding activity determined in(a) is higher than that determined in (b);

(d) linking a polynucleotide encoding the antigen-binding domain of theantibody selected in (c) to a polynucleotide encoding an FcRn-bindingdomain having FcRn-binding activity in a neutral pH range;

(e) culturing cells into which a vector to which the polynucleotideobtained in (d) is operably linked has been introduced; and

(f) collecting an antigen-binding molecule from the culture fluid of thecells cultured in (e).

In a non-limiting embodiment of the present invention, theantigen-binding domain preferably includes a plurality ofantigen-binding domains constituting an antibody fragment library.Furthermore, in a non-limiting embodiment of the present invention, theantibody preferably includes a panel of a group of antibodies that havebeen monocloned in advance. Methods for producing these libraries andantibodies are described in the “Antibodies” section mentioned above.

For example, the step of obtaining an antigen-binding domain whoseantigen-binding activity is lower under a high hydrogen ionconcentration condition or low pH, i.e. in an acidic pH range, thanunder a low hydrogen ion concentration condition or high pH, i.e. in aneutral pH range, which is a non-limiting embodiment of the presentinvention, preferably includes the step of obtaining an antigen-bindingdomain including the following steps:

(a) contacting an antigen with a library of antigen-binding domains in aneutral pH range;

(b) placing a library of the antigen-binding domains bound to theantigen in step (a) in an acidic pH range; and

(c) isolating an antigen-binding domain that is dissociated in step (b).

For example, the step of obtaining an antigen-binding domain whoseantigen-binding activity is lower under a low calcium ion concentrationcondition than under a high calcium ion concentration condition, whichis a non-limiting embodiment of the present invention, preferablyincludes the step of obtaining an antigen-binding domain including thefollowing steps:

(a) contacting an antigen with a library of antigen-binding domainsunder a high calcium ion concentration condition;

(b) placing a library of the antigen-binding domains bound to theantigen in step (a) under a low calcium ion concentration condition; and

(c) isolating an antigen-binding domain that is dissociated in step (b).

Furthermore, the step of obtaining an antigen-binding domain whoseantigen-binding activity is lower under a high hydrogen ionconcentration condition or low pH, i.e. in an acidic pH range, thanunder a low hydrogen ion concentration condition or high pH, i.e. in aneutral pH range, which is a non-limiting embodiment of the presentinvention, preferably includes the step of obtaining an antigen-bindingdomain including the following steps:

(a) contacting, in a neutral pH range, a library of antigen-bindingdomains with a column onto which an antigen has been immobilized;

(b) eluting an antigen-binding domain bound to the column in step (a)from the column in an acidic pH range; and

(c) isolating the antigen-binding domain eluted in step (b).

Furthermore, the step of obtaining an antigen-binding domain whoseantigen-binding activity is lower under a low calcium ion concentrationcondition than under a high calcium ion concentration condition, whichis a non-limiting embodiment of the present invention, preferablyincludes the step of obtaining an antigen-binding domain including thefollowing steps:

(a) contacting, under a high calcium ion concentration condition, alibrary of antigen-binding domains with a column onto which an antigenhas been immobilized;

(b) eluting an antigen-binding domain bound to the column in step (a)from the column under a low calcium ion concentration condition; and

(c) isolating the antigen-binding domain eluted in step (b).

Furthermore, the step of obtaining an antigen-binding domain whoseantigen-binding activity is lower under a high hydrogen ionconcentration condition or low pH, i.e. in an acidic pH range, thanunder a low hydrogen ion concentration condition or high pH, i.e. in aneutral pH range, which is a non-limiting embodiment of the presentinvention, preferably includes the step of obtaining an antigen-bindingdomain including the following steps:

(a) passing, in an acidic pH range, a library of antigen-binding domainsthrough a column onto which an antigen has been immobilized;

(b) collecting antigen-binding domains eluted without binding to thecolumn in step (a);

(c) allowing the antigen-binding domains collected in step (b) to bindto the antigen in a neutral pH range; and

(d) isolating an antigen-binding domain bound to the antigen in step(c).

Furthermore, the step of obtaining an antigen-binding domain whoseantigen-binding activity is lower under a low calcium ion concentrationcondition than under a high calcium ion concentration condition, whichis a non-limiting embodiment of the present invention, preferablyincludes the step of obtaining an antigen-binding domain including thefollowing steps:

(a) passing, under a low calcium ion concentration condition, a libraryof antigen-binding domains through a column onto which an antigen hasbeen immobilized;

(b) collecting antigen-binding domains eluted without binding to thecolumn in step (a);

(c) allowing the antigen-binding domains collected in step (b) to theantigen under a high calcium ion concentration condition; and

(d) isolating an antigen-binding domain bound to the antigen in step(c).

For example, the step of obtaining an antibody whose antigen-bindingactivity is lower under a high hydrogen ion concentration condition orlow pH, i.e. in an acidic pH range, than under a low hydrogen ionconcentration condition or high pH, i.e. in a neutral pH range, which isa non-limiting embodiment of the present invention, preferably includesthe step of obtaining an antibody including the following steps:

(a) determining the antigen-binding activity of an antibody in an acidicpH range;

(b) determining the antigen-binding activity of the antibody in aneutral pH range; and

(c) selecting the antibody whose antigen-binding activity determined inthe acidic pH range is lower than that determined in the neutral pHrange.

For example, the step of obtaining an antibody whose antigen-bindingactivity is lower under a low calcium ion concentration condition thanunder a high calcium ion concentration condition, which is anon-limiting embodiment of the present invention, preferably includesthe step of obtaining an antibody including the following steps:

(a) determining the antigen-binding activity of an antibody under a lowcalcium ion concentration condition;

(b) determining the antigen-binding activity of the antibody under ahigh calcium ion concentration condition; and

(c) selecting the antibody whose antigen-binding activity under the lowcalcium ion concentration condition is lower than that under the highcalcium ion concentration condition.

Furthermore, the step of obtaining an antibody whose antigen-bindingactivity is lower under a high hydrogen ion concentration condition orlow pH, i.e. in an acidic pH range, than under a low hydrogen ionconcentration condition or high pH, i.e. in a neutral pH range, which isa non-limiting embodiment of the present invention, preferably includesthe step of obtaining an antibody including the following steps:

(a) contacting an antibody with an antigen in a neutral pH range;

(b) obtaining the antibody bound to the antigen in step (a);

(c) placing the antibody obtained in step (b) in an acidic pH range; and

(d) selecting the antibody whose antigen-binding activity in step (c) isweaker than the criterion for the selection in step (b).

Furthermore, the step of obtaining an antibody whose antigen-bindingactivity is lower under a low calcium ion concentration condition thanunder a high calcium ion concentration condition, which is anon-limiting embodiment of the present invention, preferably includesthe step of obtaining an antibody including the following steps:

(a) contacting an antibody with an antigen under a high calcium ionconcentration condition;

(b) obtaining the antibody bound to the antigen in step (a);

(c) placing the antibody obtained in step (b) in a low calcium ioncondition; and

(d) selecting the antibody whose antigen-binding activity in step (c) isweaker than the criterion for the selection in step (b).

The above-mentioned steps may be repeated twice or more times.Therefore, the present invention provides antigen-binding domains orantibodies whose antigen-binding activity is lower in an acidic pH rangethan in a neutral pH range, which are obtained by the above-mentionedscreening methods which further comprise the step of repeating steps (a)to (c) or steps (a) to (d) twice or more times. The number of cycles ofsteps (a) to (c) or (a) to (d) is not particularly limited, but isusually ten or less.

In the aforementioned steps, the antigen-binding activity of anantigen-binding domain or antibody under a high hydrogen ionconcentration condition or low pH, i.e. in an acidic pH range, is notparticularly limited as long as it is antigen-binding activity at pH 4.0to 6.5, but antigen-binding activity at pH 4.5 to 6.5 may be preferred.Alternatively, antigen-binding activity at pH 5.0 to 6.5, orantigen-binding activity at pH 5.5 to 6.0 may also be preferred.

More preferred pH includes the pH in early endosomes in vivo, and aspecific example is antigen-binding activity at pH5.8. Theantigen-binding activity of an antigen-binding domain or antibody undera low hydrogen ion concentration condition or high pH, i.e. in a neutralpH range, is not particularly limited as long as it is antigen-bindingactivity at pH 6.7 to 10, but antigen-binding activity at pH 6.7 to 9.5may be preferred. Alternatively, antigen-binding activity at pH 7.0 to9.5, or antigen-binding activity at pH 7.0 to 8.0 may also be preferred.More preferred pH includes the pH in blood plasma in vivo, and aspecific example is antigen-binding activity at pH7.4.

In the aforementioned steps, the antigen-binding activity of anantigen-binding domain or antibody under a low calcium concentrationcondition is not particularly limited as long as it is antigen-bindingactivity at an ionized calcium concentration of 0.1 μM to 30 μM, butantigen-binding activity at an ionized calcium concentration of 0.2 μMto 20 μM may be preferred. In another embodiment, antigen-bindingactivity at 0.5 μM to 10 μM may be preferred. More preferred ionizedcalcium concentrations include the ionized calcium concentration inearly endosomes in vivo, and specific examples include antigen-bindingactivity at 1 μM to 5 μM and antigen-binding activity at 2 μM to 4 μM.The antigen-binding activity of an antigen-binding domain or antibodyunder a high calcium concentration condition is not particularly limitedas long as it is antigen-binding activity at an ionized calciumconcentration of 100 μM to 10 mM, but antigen-binding activity at anionized calcium concentration of 200 μM to 5 mM may be preferred. In adifferent embodiment, antigen-binding activity at 500 μM to 2.5 mM maybe preferred, and in another embodiment, antigen-binding activity at 200μM to 2 mM may be preferred. In a different embodiment, antigen-bindingactivity at 400 μM to 1.5 mM may also be preferred. More preferredionized calcium concentrations include the ionized calcium concentrationin plasma in vivo, and specific examples includes antigen-bindingactivity at 0.5 mM to 2.5 mM.

FcRn-binding domains having FcRn-binding activity in a neutral pH range,modified Fc regions with Fcγ-receptor-binding activity that is higherthan the Fcγ-receptor-binding activity of a naturally-occurring Fcregion in which fucose is attached to the sugar chain at position 297according to EU numbering, and Fc regions including such FcRn-bindingdomains and modified Fc regions are obtained by methods described in thesection “FcRn-binding domains” and “FcRn-binding domains” mentionedabove. Polynucleotides encoding each of the domains may be obtained byknown genetic recombination methods described later in this section.

Respective domains of the present invention can be linked together vialinkers or directly via polypeptide binding. The linkers comprisearbitrary peptide linkers that can be introduced by genetic engineering,synthetic linkers, and linkers disclosed in, for example, ProteinEngineering (1996) 9(3), 299-305. However, peptide linkers are preferredin the present invention. The length of the peptide linkers is notparticularly limited, and can be suitably selected by those skilled inthe art according to the purpose. The length is preferably five aminoacids or more (without particular limitation, the upper limit isgenerally 30 amino acids or less, preferably 20 amino acids or less),and particularly preferably 15 amino acids.

For example, such peptide linkers preferably include:

Ser Gly⋅Ser Gly⋅Gly⋅Ser Ser⋅Gly⋅Gly Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 29)Ser⋅Gly⋅Gly⋅Gly (SEQ ID NO: 30) Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 31)Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 32) Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO:33) Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 34) Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser(SEQ ID NO: 35) Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 36)(Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 31))n (Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO:32))n

where n is an integer of 1 or larger. The length or sequences of peptidelinkers can be selected accordingly by those skilled in the artdepending on the purpose.

Synthetic linkers (chemical crosslinking agents) is routinely used tocrosslink peptides, and for example:

N-hydroxy succinimide (NHS),disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidyl propionate) (DSP),dithiobis(sulfosuccinimidyl propionate) (DTSSP),ethylene glycol bis(succinimidyl succinate) (EGS),ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES),and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).These crosslinking agents are commercially available.

When multiple linkers for linking the respective domains are used, theymay all be of the same type, or may be of different types.

In addition to the linkers exemplified above, linkers with peptide tagssuch as His tag, HA tag, myc tag, and FLAG tag may also be suitablyused. Furthermore, hydrogen bonding, disulfide bonding, covalentbonding, ionic interaction, and properties of binding with each other asa result of combination thereof may be suitably used. For example, theaffinity between CH1 and CL of antibody may be used, and Fc regionsoriginating from the above-described bispecific antibodies may also beused for hetero Fc region association. Moreover, disulfide bonds formedbetween domains may also be suitably used.

In order to link respective domains via peptide linkage, polynucleotidesencoding the domains are linked together in frame. Known methods forlinking polynucleotides in frame include techniques such as ligation ofrestriction fragments, fusion PCR, and overlapping PCR. Such methods canbe appropriately used alone or in combination to constructantigen-binding molecules of the present invention.

Known methods may be appropriately employed for isolatingpolynucleotides encoding each domain. For example, a polynucleotidesequence encoding an antigen-binding domain is isolated from a phagedisplaying an antigen-binding domain of interest from a librarycontaining a plurality of antigen-binding domains, by PCR using primersused for constructing the library or primers having the sequence of thephage vector used for constructing the library. In order to obtain genesencoding the antibody variable regions, it is convenient to use the5′-RACE method using primers for amplifying the variable region genes.First, RNAs extracted from hybridoma cells are used as templates tosynthesize cDNAs and thereby obtain a 5′-RACE cDNA library. Commerciallyavailable kits such as SMART RACE cDNA amplification kit areappropriately used for synthesis of a 5′-RACE cDNA library.

The obtained 5′-RACE cDNA library is used as a template to amplifyantibody genes by PCR. Primers for amplifying mouse antibody genes maybe designed based on known antibody gene sequences. The nucleotidesequences of these primers vary depending on the subclass ofimmunoglobulin. Therefore, it is preferred to determine the subclass inadvance using a commercially available kit such as Iso Strip mousemonoclonal antibody isotyping kit (Roche Diagnostics).

More specifically, when acquisition of genes encoding mouse IgG isintended, primers capable of amplifying genes encoding γ1, γ 2a, γ 2b,and γ 3 for the heavy chain, and genes encoding the κ chain and λ chainfor the light chain, may be used. To amplify the genes of variableregions of IgG, generally, a primer that anneals to a portioncorresponding to the constant region close to the variable region isused as the 3′ primer. On the other hand, a primer supplied in the5′-RACE cDNA library production kit can be used as the 5′ primer.

After the polynucleotide sequence of the antigen-binding domain orantibody of the present invention isolated as described above isdetermined, a polynucleotide containing a fused gene in which thispolynucleotide is linked in frame with a polynucleotide encoding anFcRn-binding domain having FcRn-binding activity in a neutral pH rangeis produced. The produced polynucleotide containing the fused gene isoperably linked to a suitable expression vector so that it will beexpressed in desired cells.

The terms “cell”, “cell line”, and “cell culture” are used synonymouslyherein, and such designations may include all progeny of a cell or cellline. Thus, for example, the terms “transformants” and “transformedcells” include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content due todeliberate or inadvertent mutations. Mutant progeny that havesubstantially the same function or biological activity as screened forin the originally transformed cell may also be included. Where distinctdesignations are intended, such intention will be clear from the contextof the description.

When referring to the expression of a coding sequence, the term “controlsequences” refers to DNA nucleotide sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotesinclude, for example, a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers for the expression of a codingsequence.

For a nucleic acid, the term “operably linked” means that the nucleicacid is placed into a functional relationship with another nucleic acidsequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as aprecursor protein that participates in the secretion of the polypeptide.A promoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. A ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading frame. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at suitablerestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice. Furthermore, linked nucleic acids may be producedby the above-mentioned overlap extension PCR technique.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo nucleic acid fragments. For ligation of the two fragments, the endsof the fragments must be compatible with each other. In some cases, theends will be directly compatible after endonuclease digestion. However,it may be necessary first to convert the staggered ends commonlyproduced after endonuclease digestion to blunt ends to make themcompatible for ligation. For blunting the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with about 10 units ofthe Klenow fragment of DNA polymerase I or T4 DNA polymerase in thepresence of the four deoxyribonucleotide triphosphates. The DNA is thenpurified by phenol-chloroform extraction and ethanol precipitation, orby silica purification. The DNA fragments that are to be ligatedtogether are put in solution in equimolar amounts. The solution willcontain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about10 units per 0.5 μg of DNA. If the DNA is to be ligated into a vector,the vector is first linearized by digestion with the appropriaterestriction endonuclease(s). The linearized fragment is then treatedwith bacterial alkaline phosphatase or calf intestinal phosphatase toprevent self-ligation of the fragment during the ligation step.

Production of an expression vector containing a polynucleotide encodingan antigen-binding molecule of the present invention, introduction ofthe expression vector into cells, expression of the polynucleotide inthe cells, and acquisition of the expressed antigen-binding moleculefrom the culture fluid of the cells are carried out according to themethods described in the “Antibodies” section mentioned above.

All prior art references cited in the present specification are hereinincorporated by reference.

EXAMPLES

Herein below, the present invention will be specifically described withreference to the Examples, but it is not to be construed as beinglimited thereto.

[Example 1] Production of a Mouse Model that is Immune Tolerant to HumanIL-6 Receptor

Since the IL-6 receptor is highly expressed in myeloma, for which thegrowth factor is the ligand IL-6, anti-human IL-6 receptor antibodiesare known to exhibit antitumor effects in an immunodeficient mousexenograft model of human myeloma (Eur. J. Immunol. 22, 1989-93 (1992)).If mouse anti-IL-6 receptor antibody production can be induced byadministering anti-IL-6 receptor antibodies to mice, those anti-IL-6receptor antibodies may induce acquired immunity and may be shown to beuseful as cancer vaccines.

However, since the mouse immune functions do not work in immunodeficientmice that are used as the antitumor models, inducing acquired immunityis impossible. Even if soluble human IL-6 receptors are administered tonormal mice, mouse-anti-human IL-6 receptor antibodies will be quicklyproduced against the human IL-6 receptors which are foreign substancesfor the mice. Therefore, normal mice cannot be used as they are assystems for evaluating effects of anti-human IL-6 receptor antibodies onthe production of mouse anti-human IL-6 receptor antibodies (acquiredimmunity to the human IL-6 receptor).

On the other hand, in clinical situations in humans, since the antigentargeted by the antibody molecules is a human antigen, humans areimmunotolerant toward that targeted self-antigen. In clinical situationsin humans, when production of autoantibodies against a target antigen isinduced following administration of antibodies against the targetantigen, immunotolerance is presumably lost.

Therefore, for an evaluation envisioning the clinical situation inhumans regarding whether the antibodies administered to mice can induceacquired immunity against the target human antigen in the mice, it isnecessary that the mice is in a state close to immunotolerance againstthe target human antigen (simply administering a target human antigen tomice would not lead to production of antibodies against the target humanantigen by the mice), and it is necessary to establish an assay systemfor evaluating that, upon administration of antibodies against thetarget antigen, the near-immunotolerant state is lost and production ofautoantibodies against the target antigen is induced.

Therefore, to avoid production of autoantibodies against the targethuman antigen in the mice by administration of the target human antigento the mice, an assay system was constructed, in which CD4-positive Tcells necessary for inducing antibody production from antigenpresentation were sufficiently removed using anti-mouse CD4 antibodies.

The following test model was constructed as a model for maintaining theplasma concentration of the soluble human IL-6 receptor, the targethuman antigen, at a constant state (approximately 20 ng/mL). An infusionpump (MINI-OSMOTIC PUMP MODEL 2004; alzet) filled with soluble humanIL-6 receptor was implanted under the skin on the back of normal mice(C57BL/6J mouse, Charles River Japan) to prepare model animals in whichthe plasma concentration of soluble human IL-6 receptor was kept in asteady state.

The study was conducted in two groups (n=4 per group). To the group ofmice that mimic immune tolerance, a single dose (20 mg/kg) of monoclonalanti-mouse CD4 antibody (R&D) was administered into the caudal vein toinhibit the production of mouse antibodies against soluble human IL-6receptor. Subsequently, the antibody was similarly administered once in10 days (hereinafter referred to as anti-mouse CD4 antibodyadministration group). The other group was used as a control group,i.e., anti-mouse CD4 antibody non-administration group that received nomonoclonal anti-mouse CD4 antibody. Subsequently, an infusion pumpfilled with 92.8 μg/mL soluble human IL-6 receptor was subcutaneouslyimplanted into the back of a mouse. After the implantation of aninfusion pump, blood samples were collected over time, immediatelyfollowed by centrifugation for 15 minutes at 4° C. and 15,000 rpm toobtain plasma. The separated plasma was stored in a freezer set to −20°C. or lower until the time of measurement. The plasma concentration ofsoluble human IL-6 receptor (hsIL-6R) was determined by the methoddescribed below.

hsIL-6R concentration in mouse plasma was determined usingelectrochemiluminescence method. An hsIL-6R calibration curve sampleprepared at 2,000, 1,000, 500, 250, 125, 62.5, or 31.25 pg/mL, and amouse plasma measurement sample diluted by 50-fold or above, were mixedwith a monoclonal anti-human IL-6R antibody (R&D) ruthenated withSULFO-TAG NHS Ester (Meso Scale Discovery), a biotinylated anti-humanIL-6R antibody (R&D), and tocilizumab, followed by overnight reaction at37° C. Tocilizumab was prepared at a final concentration of 333 μg/mL.Subsequently, the reaction liquid was dispensed into an MA400 PRStreptavidin Plate (Meso Scale Discovery). In addition, after washingthe reaction liquid that was allowed to react at room temperature for 1hour, Read Buffer T (×4) (Meso Scale Discovery) was dispensed.Subsequently, the reaction liquid was immediately subjected tomeasurement using a SECTOR PR 400 Reader (Meso Scale Discovery). Theconcentration of hsIL-6R was calculated from the response of thecalibration curve using the SOFTmax PRO analysis software (MolecularDevices). The change in plasma hsIL-6R concentration of each of thenormal mice, which was determined by this method, is shown in FIG. 1.

As a result, in all mice of the anti-mouse CD4 antibody non-administeredgroup, a decrease in the plasma hsIL-6R concentration was observed 14days after implantation of the infusion pump under the skin on the backof the mice. That is, if mouse anti-human IL-6 receptor antibodies areproduced, the plasma hsIL-6R concentration will decrease, and thisshowed that mouse anti-human IL-6 receptor antibodies are produced inthese groups. On the other hand, a decrease in the plasma hsIL-6Rconcentration was not observed in all mice belonging to the anti-mouseCD4 antibody-administered group. Thus, this showed that mouse anti-humanIL-6 receptor antibodies were not produced since, throughout the 28 daysduring which the infusion pump was effective, a nearly constant plasmahsIL-6R concentration (approximately 20 ng/mL) has been maintained.

The above results confirmed that prior administration of anti-mouse CD4antibody does not lead to induction of acquired immunity in normal micewhen the human IL-6 receptor, which is the target antigen, isadministered alone. In the Examples that follow, the anti-mouse CD4antibody-administered model was used as the system for evaluating theinduction of acquired immunity toward human IL-6 receptor followinganti-human IL-6 receptor antibody administration.

[Example 2] Comparison of the Acquired Immunity-Inducing Effects byNormal Anti-IL-6 Receptor Antibodies and pH-Dependent Anti-Human IL-6Receptor Antibodies in Normal Mouse Model with Immune Tolerance for theHuman IL-6 Receptor

For induction of acquired immunity to a target antigen, the targetantigen taken up into antigen-presenting cells must be appropriatelydegraded by lysosomes in the cells, and fragmented peptides of thetarget antigen must undergo antigen presentation by binding to MHC classI or MHC class II. It is considered that the larger the number ofpeptides presented as antigens, the stronger the induction of immunity;thus, as a method for enhancing induction of acquired immunity to thetarget antigen, a method for sending a larger number of the targetantigen into the antigen-presenting cells was considered.

Ordinarily, when an antigen is incorporated non-specifically into anantigen-presenting cell, it is transferred as is from the endosome tothe lysosome, and therefore, fragmented peptides may be presented asantigens. However, when an ordinary IgG antibody is bound to an antigen,since the IgG antibody is recycled to the cell surface (in plasma) fromthe inside of the endosome by binding to FcRn, the antigen bound to theantibody may not be transferred to the lysosome and may be recycled tothe cell surface (in plasma). Therefore, administration of normal IgGantibodies will suppress degradation of the target antigen. As a result,fragmented peptides of the target antigen which were appropriatelydegraded in the lysosome in the antigen-presenting cells are notpresented as antigens, and rather, induction of acquired immunityagainst the target antigen is thought to decrease. Accordingly, sinceIgG antibodies having a pH-dependent binding activity, such that theybind to the target antigen at pH7.4 in the plasma and release theantigen under acidic conditions of pH5.0 to pH6.5 in the endosome (WO2009/125825), can dissociate the target antigen in the acidic endosomesof the antigen-presenting cells, it was considered that by using thisIgG antibody, only the target antigen may translocate into the lysosomesof the antigen-presenting cells, the fragmented peptides of the targetantigen may be presented as antigens, and the IgG antibodies thatreleased the antigen may be recycled to the cell surface (in plasma)from inside the endosomes by binding to FcRn without being subjected toantigen presentation.

Therefore, the acquired immunity-inducing effects of normal anti-humanIL-6 receptor IgG antibodies and of pH-dependent anti-human IL-6receptor IgG antibodies were assessed using the normal mouse model thatis immune tolerant to human IL-6 receptors, established in Example 1.H54/L28-IgG1 composed of H54-IgG1 (SEQ ID NO: 38) and L28-CK (SEQ ID NO:39), which is described in WO 2009/125825, was used as the ordinaryanti-human IL-6 receptor IgG antibody. Fv4-IgG1 composed of VH3-IgG1(SEQ ID NO: 17) and VL3-CK (SEQ ID NO: 40), was used as the pH-dependentanti-human IL-6 receptor IgG antibody. Antibody preparation was carriedout using the method indicated in Reference Example 1.

The acquired immunity-inducing effects of H54/L28-IgG1 and Fv4-IgG1 wereassessed using the human IL-6 receptor-immunotolerant mouse modelestablished from human FcRn transgenic mice (B6.mFcRn−/−.hFcRn Tg line32+/+ mouse, Jackson Laboratories (Methods Mol. Biol. (2010) 602:93-104)). H54/L28-IgG1 and Fv4-IgG1 were administered to the human IL-6receptor-immunotolerant mouse models. Specifically, in a similar mannerto Example 1, three days after infusion pump implantation, anti-humanIL-6 receptor antibody was administered once at 1 mg/kg into the tailvein. Blood was collected over time after administration of theanti-human IL-6 receptor antibody. The collected blood was immediatelycentrifuged at 15,000 rpm and 4° C. for 15 minutes to obtain the plasma.The separated plasma was stored in a freezer set at −20° C. or loweruntil performing the measurements. The plasma hsIL-6R concentration wasmeasured by the same method as that described in Example 1.

The changes in the mean plasma hsIL-6R concentration for each of thecontrol (anti-human IL-6 receptor antibody non-administered group),H54/L28-IgG1-administered, and Fv4-IgG1-administered groups are shown inFIG. 2. In all animals of the control group, the plasma hsIL-6Rconcentration was maintained at a nearly constant level as in Example 1,indicating that mouse anti-human IL-6 receptor antibodies are notproduced. A significant elevation in the plasma hsIL-6R concentrationwas observed in all animals of the H54/L28-IgG1-administered group ascompared with the control, and this elevated state continued for 28days. This was thought to be because, when hsIL-6R is taken upnonspecifically into cells, it is directly degraded in the lysosomes,whereas hsIL-6R bound to an IgG antibody is recycled to the plasma as anantibody-antigen complex by FcRn, so that hsIL-6R clearance is reducedand consequently the plasma hsIL-6R concentration increases. Since thiselevated state continued for 28 days, this indicated that in a similarmanner to the control, mouse anti-human IL-6 receptor antibodyproduction is not induced in the H54/L28-IgG1-administered group. In allanimals of the Fv4-IgG1-administered group, the plasma hsIL-6Rconcentration increased as compared to that of the control, but thedegree of elevation of the plasma hsIL-6R concentration was clearlyreduced as compared to that of the H54/L28-IgG1-administered group. Thisis because the antibody with pH-dependent binding releases the antigenin the endosome, leading to suppression of reduction of hsIL-6Rclearance, which then leads to suppression of elevation of plasmahsIL-6R concentration. It was considered from these results that thepH-dependently binding antibody releases more target antigens in theendosomes of antigen-presenting cells as compared with normalantibodies, and promotes translocation of the target antigen to thelysosomes. However, since the state of elevated hsIL-6R concentrationwas maintained for 28 days, in a similar manner to H54/L28-IgG1, it wasshown that Fv4-IgG1 does not induce the production of mouse anti-humanIL-6 receptor antibodies.

These results revealed that the acquired immunity cannot be inducedagainst the target antigen using ordinary IgG antibodies and usingpH-dependent binding IgG antibodies which release the antigen inside theendosomes of antigen-presenting cells for transfer to the lysosomes.

[Example 3] Effects of pH-Dependent IL-6 Receptor Binding andEnhancement of FcRn Binding at pH7.4 on Acquired Immunity-InducingEffects in Human FcRn Transgenic Mouse Model with Immune Tolerance forthe Human IL-6 Receptor (3-1) Summary

In Example 2, it was confirmed that an ordinary IgG1 antibody and anIgG1 antibody with pH-dependent binding cannot induce acquired immunityagainst a target antigen. On the other hand, as a method for enhancingthe immunogenicity of a T-cell epitope peptide, a method of fusing theT-cell epitope peptide with Fc and then enhancing the binding of the Fcportion to FcRn at pH 7.4 so that more T-cell epitope peptides aretransferred to lysosomes, has been reported recently (J. Immunol. 181,7550-61 (2008)). Since FcRn is expressed on antigen-presenting cells,enhancing binding of the Fc portion to FcRn at pH7.4 may promote antigenpresentation of the T-cell epitope peptide. However, since the moleculedisclosed in this method, which has an antigenic peptide directly fusedto Fc, cannot bind to cancer antigens as an antigen-binding molecule,this molecule cannot exhibit any direct action on cancer cells.Furthermore, the method for enhancing FcRn-binding of the Fc portion atpH7.4 enhances immunogenicity of the T-cell epitope peptide in vitro,but on the contrary, decreases immunogenicity in vivo, and this was noteffective in vivo. This way, since the molecule produced by directlyfusing a target antigen with the Fc having enhanced FcRn binding atpH7.4 cannot show a binding activity toward a target antigen, it cannotact directly on a target antigen, and moreover, enhancing FcRn-bindingin vivo resulted in decreasing the immunogenicity

Taking into account reports made so far and Example 2, an antibody(H54/L28-F157) produced by introducing to a normal IgG1 antibody(H54/L28-IgG1) alterations that enhance FcRn binding at pH7.4, and anantibody (Fv4-F157) produced by introducing to the IgG1 antibody withpH-dependent binding (Fv4-IgG1) alterations that enhance FcRn binding atpH7.4 were administered to assess whether acquired immunity can beinduced toward the target antigen.

(3-2) Antibody Production

H54/L28-F157 composed of H54-F157 (SEQ ID NO: 41) and L28-CK (SEQ ID NO:39) was used as the normal anti-human IL-6 receptor IgG antibody withenhanced FcRn binding. Fv4-F157 composed of VH3-F157 (SEQ ID NO: 42) andVL3-CK (SEQ ID NO: 40) was used as the pH-dependent anti-human IL-6receptor IgG antibody with enhanced FcRn binding. Antibody preparationwas carried out using the method of Reference Example 1.

(3-3) Measurement of Affinity to Human FcRn

To determine the affinities of the Fc regions of Fv4-IgG1 and Fv4-F157(referred to as IgG1 and F157, respectively) produced in Example 2 tohuman FcRn at pH7.0, the affinities of VH3/L(WT)-IgG1 composed ofVH3-IgG1 (SEQ ID NO: 17) and L(WT)-CK (SEQ ID NO: 18) and VH3/L(WT)-F157composed of VH3-F157 (SEQ ID NO: 42) and L(WT)-CK (SEQ ID NO: 18) tohuman FcRn were determined by the method shown below.

Kinetic analyses between human FcRn and the antibody were carried outusing Biacore T100 (GE Healthcare). A suitable amount of protein L(ACTIGEN) was immobilized onto a Sensor chip CM4 (GE healthcare) by theamine coupling method, and antibodies of interest were captured onto it.Next, diluted solutions of FcRn and the running buffer which is theblank were injected, and human FcRn was made to interact with theantibodies captured on the sensor chip. The running buffer used was 50mmol/L sodium phosphate, 150 mmol/L NaCl, 0.05% (w/v) Tween 20, pH7.0,and the buffer was used for the FcRn dilutions. The sensor chip wasregenerated using 10 mmol/L Glycine-HCl, pH1.5. All measurements werecarried out at 25° C. Based on the association rate constant ka (1/Ms)and dissociation rate constant kd (1/s), which are kinetic parameters,calculated from the sensorgram obtained by the measurement, the KD (M)(affinity) of each antibody toward human FcRn was calculated. Eachparameter was calculated using the Biacore T100 Evaluation Software (GEHealthcare).

The affinities of IgG1 and F157 to human FcRn are shown in Table 7. Theaffinity of F157 to human FcRn was confirmed to be approximately600-fold higher as compared with IgG1.

TABLE 7 Affinity to human FcRn IgG1 8.8E−05 F157 1.5E−07

(3-4) Change in the Plasma Concentration of the Antigen (Soluble HumanIL-6 Receptor) in the Antibody Administration Test

Next, in Test 1, the mouse model with immune tolerance for the humanIL-6 receptor which was established from human FcRn transgenic mice(B6.mFcRn−/−.hFcRn Tg line 32+/+ mouse, Jackson Laboratories, MethodsMol. Biol. (2010) 602, 93-104) was used. H54/L28-F157 and Fv4-F157 wereadministered to the human IL-6 receptor-immunotolerant mouse models.Specifically, in a similar manner to Example 1, three days afterimplantation of the infusion pump, an anti-human IL-6 receptor antibodywas administered once at a dose of 1 mg/kg into the tail vein (threeindividuals in each group). Blood was collected over time afteradministration of the anti-human IL-6 receptor antibody to the mice.Furthermore, in Test 2, blood was collected over time afteradministration of Fv4-F157 alone in a similar manner. The collectedblood was immediately centrifuged at 15,000 rpm and 4° C. for 15 minutesto obtain the plasma. The separated plasma was stored in a freezer at−20° C. or lower until performing the measurements. The plasma hsIL-6Rconcentration was measured by the same method as that described inExample 1.

The changes in the mean plasma hsIL-6R concentration for each of theH54/L28-F157-administered and Fv4-F157-administered groups of Test 1 andthe Fv4-F157-administered group of Test 2 are shown in FIG. 3, togetherwith the change in the mean plasma hsIL-6R concentration for each of thecontrol (antibody non-administration group), theH54/L28-IgG1-administered and Fv4-IgG1-administered groups obtained inExample 2. The H54/L28-F157-administered group showed an increase inplasma hsIL-6R concentration in a similar manner to theH54/L28-IgG1-administered group, but the increase was transient, and 13days later, the plasma hsIL-6R concentration decreased to a levelequivalent to that of the control. Thereafter, a level equivalent tothat of the control was maintained for 28 days; therefore, in the samemanner as H54/L28-IgG1, mouse anti-human IL-6 receptor antibodies wereshown not to be produced, and it was revealed that enhancement ofFcRn-binding at pH7.4 alone cannot induce acquired immunity toward thetarget antigen. This does not conflict with the finding that a moleculeproduced by fusing a T-cell epitope peptide to the Fc region withenhanced FcRn-binding at pH7.4 cannot enhance induction of acquiredimmunity against the T-cell epitope peptide, as reported in J. Immunol.(2008), 181, 7550-7561. In contrast, in the Fv4-F157-administered group,a rapid reduction of the plasma hsIL-6R concentration was observed afterantibody administration, and one day after administration, the hsIL-6Rconcentration decreased to below detection limit (1.56 ng/mL or lower).

Without being limited to a particular theory, the above-mentionedphenomena may be described as follows. Enhancement of acquired immunityto a target antigen may depend on the amount of the target antigen takenup into antigen-presenting cells by the administered antibody. Theamount of the incorporated target antigen can be evaluated bydetermining how much the concentration of the target antigen in theplasma decreased. Although the antibody with pH-dependent binding(Fv4-IgG1) releases the target antigen in the acidic endosome afterbeing taken up into cells, the process of incorporation into cells ofthe pH-dependent binding antibody that is bound to the target antigen isdependent on non-specific pinocytosis, and since the speed ofincorporation is slow, the concentration of the target antigen in theplasma does not decrease to a level lower than that of the control (FIG.4). Furthermore, the antibody (H54/L28-F157) in which only the FcRnbinding at pH7.4 is enhanced is aggressively taken up into cells usingthe FcRn-binding at pH7.4; however, since most of it is recycleddirectly to the cell surface by the intrinsic function of FcRn in astate where the target antigen is still bound to the antibody, that is,as a complex of the antibody and the target antigen, the concentrationof the target antigen in the plasma does not decrease to a level belowthat of the control (FIG. 5). In contrast, since the antibody withpH-dependent binding with enhanced FcRn-binding at pH7.4 (Fv4-F157)which was aggressively taken up into cells through FcRn-binding at pH7.4releases the target antigen in the endosome, the antigen is sent to thelysosome, whereas the antibody that released the target antigen isrecycled to the cell surface by the intrinsic function of FcRn, bindsagain to a target antigen, and can similarly send the target antigenagain to the lysosome (FIG. 6). It was considered that only theantibodies with pH-dependent binding that have enhanced FcRn-binding atpH7.4 are able to reduce the concentration of the target antigen in theplasma to a level that is significantly lower than that of the controlby repeating this cycle.

The plasma hsIL-6R concentration for each of the sixFv4-F157-administered mice of 1 and 2 are shown in FIGS. 7 and 8. Amongthe six Fv4-F157-administered mice, the hsIL-6R concentration increasedafter day 13 in three mice, namely #7, #8, and #10, then returned to thelevel equivalent to that of the control, and thereafter, the levelequivalent to that of the control was maintained for 28 days. On theother hand, in the remaining three mice, namely #9, #11, and #12, sincethe hsIL-6R concentration did not recover and maintained thereafter thatlow value for up to 28 days, it was considered that in these three mice,mouse anti-human IL-6 receptor antibodies were produced and wereremoving the human IL-6 receptors from within the plasma. Therefore, themouse anti-human IL-6 receptor antibody titer was determined in the sixFv4-F157-administered mice by the method indicated below.

(3-4) Change in Antibody Titer of the Mouse Antibody (Mouse Anti-HumanIL-6 Receptor Antibody) Against an Antigen (Soluble Human IL-6 Receptor)in Mice in an Antibody Administration Test

The titer of the mouse anti-human IL-6 receptor antibody in the mouseplasma was measured by electrochemiluminescence. First, human IL-6receptor was dispensed into MA100 PR Uncoated plate (Meso ScaleDiscovery), and by letting it stand overnight at 4° C., a human IL-6receptor-immobilized plate was produced. Mouse plasma measurementsamples diluted 50-fold were dispensed into the human IL-6receptor-immobilized plate, and this was left to stand overnight at 4°C. Then, the plate on which anti-mouse IgG (whole molecule)(Sigma-Aldrich) that was ruthenium-labeled using SULFO-TAG NHS Ester(Meso Scale Discovery) was allowed to react at room temperature for onehour and then washed. After dispensing Read Buffer T (×4) (Meso ScaleDiscovery) into the plate, measurements were taken immediately on aSECTOR PR 400 reader (Meso Scale Discovery).

The changes in mouse anti-human IL-6 receptor antibody (anti-hsIL-6Rantibody) titer in the Fv4-F157-administered group are shown in FIGS. 7and 8. As a result, in the three mice, #7, #8, and #10, whose hsIL-6Rconcentration returned to the same level as that of the control,elevation of mouse anti-human IL-6 receptor antibody titer was notobserved; however, in the three mice, #9, #11, and #12, which maintainedlow levels of hsIL-6R concentration until Day 28, elevation of mouseanti-human IL-6 receptor antibody (anti-hsIL-6R antibody) titer wasobserved. From the above-mentioned results, administration of Fv4-F157,which is a pH-dependent anti-human IL-6 receptor IgG antibody withenhanced FcRn-binding, to a normal mouse model with immune tolerance forthe human IL-6 receptor was found to be able to induce acquired immunityto the human IL-6 receptor, which is the target antigen in three out ofsix cases. Accordingly, even if the normal IgG antibody (H54/L28-IgG1),or the IgG antibody (Fv4-IgG1) showing pH-dependent binding to thetarget antigen, or the IgG antibody (H54/L28-IgG1) with enhancement ofonly the FcRn-binding at pH7.4 is administered in vivo, acquiredimmunity to the target antigen cannot be induced, and only the IgGantibody (Fv4-F157) showing pH-dependent target-antigen-binding andenhanced FcRn-binding at pH7.4 was found to be able to induce acquiredimmunity to the target antigen.

(3-5) Change in Antibody Titer of the Mouse Antibody (MouseAnti-Fv4-F157 Antibody) Against the Antibody (Fv4-F157) Administered toMice in an Antibody Administration Test

Even if production of antibodies against the target antigen becomespossible by the above-mentioned method (J. Immunol. (2008) 181,7550-7561) that uses as a pharmaceutical agent a molecule produced bydirectly fusing the target antigen to Fc with enhanced FcRn-binding atpH7.4,

sin

Affinity to human FcRn IgG1 8.8E−05 F157 1.5E−07ce antibodies against the target antigen will bind to the pharmaceuticalagent itself, they will act as anti-pharmaceutical-agent antibodies, andwill lead to reduction of the action of the pharmaceutical agent.Therefore, using a molecule having the target antigen directly fused tothe pharmaceutical agent (for example, the compounds described in J.Immunol. (2008) 181, 7550-7561 and J. Immunol. (2011), 186, 1218-1227)means inducing production of antibodies against the target antigen, ormore specifically, inducing anti-pharmaceutical-agent antibodies againstthe pharmaceutical agent itself which will lead to reduction of theaction of the pharmaceutical agent, and therefore this use may not befavorable.

The pH-dependent anti-human IL-6 receptor IgG antibody (Fv4-F157) withenhanced binding to FcRn is a pharmaceutical agent having pH-dependentbinding activity to the human IL-6 receptor, which is the targetantigen, and is not a molecule with a directly fused target antigen.Therefore, as shown in FIGS. 7 and 8, while Fv4-F157 induced antibodyproduction against a human IL-6 receptor, which is the target antigen,it may not have produced anti-pharmaceutical-agent antibodies againstthe pharmaceutical agent itself (Fv4-F157). Therefore, the mouseanti-Fv4-F157 antibody titers in the six Fv4-F157-administered mice weredetermined by the method indicated below.

The anti-Fv4-F157 antibody titer in mouse plasma was measured byelectrochemiluminescence. First, anti-human IL-6 receptor antibody wasdispensed into MA100 PR Uncoated Plate (Meso Scale Discovery), and byletting it stand overnight at 4° C., an anti-human IL-6receptor-immobilized plate was produced. Mouse plasma measurementsamples diluted 50-fold were dispensed into the anti-human IL-6 receptorantibody-immobilized plate, and this was left to stand overnight at 4°C. Then, the plate on which anti-mouse IgG (whole molecule)(Sigma-Aldrich) that was ruthenium-labeled using SULFO-TAG NHS Ester(Meso Scale Discovery) was allowed to react at room temperature for onehour, and then washed. After dispensing Read Buffer T (×4) (Meso ScaleDiscovery) into the plate, measurements were taken immediately on aSECTOR PR 400 reader (Meso Scale Discovery).

The changes in mouse anti-Fv4-F157 antibody titer and mouse anti-humanIL-6 receptor antibody (anti-hsIL-6R antibody) titer in theFv4-F157-administered group are shown in FIGS. 9 and 10. In the Fv4-F157administration group, regardless of the production of mouse anti-humanIL-6 receptor antibody (anti-hsIL-6R antibody), production of mouseanti-Fv4-F157 antibody was not observed in any mouse. Accordingly, sincethe pH-dependent anti-human IL-6 receptor IgG antibody with enhancedFcRn-binding at pH7.4 (Fv4-F157) induced production of antibodiesagainst the target antigen (human IL-6 receptor) but did not induceproduction of antibodies against the pharmaceutical agent itself(Fv4-F157), the pH-dependent (anti-human IL-6 receptor) IgG antibodywith enhanced FcRn-binding at pH7.4 was considered to be very useful asa pharmaceutical agent for inducing acquired immunity toward the targetantigen.

So far, as a method for inducing acquired immunity to a cancer antigen,the method of using a pharmaceutical agent molecule in which a cancerantigen against which one wants to induce acquired immunity is fusedwith an antibody that binds to a receptor expressed onantigen-presenting cells has been reported (J. Immunol. (2011) 186,1218-1227). Such a pharmaceutical agent molecule cannot directly bind tocancer antigens and exhibit actions (FIG. 11). Therefore, such apharmaceutical agent molecule cannot show direct cytotoxic activitytoward cancer cells or effects of inhibiting the functions of cancerantigens similarly to conventional antibody pharmaceuticals.Furthermore, since such a pharmaceutical agent molecule is incorporatedinto antigen-presenting cells as a whole and then degraded, fragmentedpeptides of the pharmaceutical agent molecule are presented on MHC classII and MHC class I. Therefore, not only cellular immunity and humoralimmunity against cancer antigens, but also humoral immunity against thepharmaceutical agent itself may also be induced, and may readily lead toreduction of effects by production of anti-pharmaceutical-agentantibodies. Therefore, such a molecule is considered unfavorable (FIG.11).

In contrast, the antibody discovered in the present Examples, whichshows pH-dependent binding to the target cancer antigen and enhancedFcRn-binding at pH7.4 can exhibit actions of binding directly to cancerantigens (FIG. 12). Therefore, it can show direct cytotoxic activitytoward cancer cells and effects of inhibiting the functions of cancerantigens similarly to conventional antibody pharmaceuticals.Furthermore, such an antibody releases the target cancer antigen in theacidic endosome and the antibody itself is recycled to the cell surfaceso that the target cancer antigen is selectively degraded, and sincepeptide fragments of the target antigen are presented on MHC class IIand MHC class I, production of anti-pharmaceutical-agent antibodiestoward the antibody itself is not induced, and cellular immunity andhumoral immunity can be induced selectively against cancer antigens(FIG. 12).

[Example 4] Effects of pH-Dependent IL-6 Receptor Binding andEnhancement of FcRn Binding at pH7.4 on Effects of Inducing AcquiredImmunity Against the Endogenous Human IL-6 Receptor in Human IL-6Receptor-Knock-In Mice (4-1) Summary

As shown in Example 3, the antibody (H54/L28-F157) produced byintroducing to a normal IgG1 antibody (H54/L28-IgG1) alterations thatenhance FcRn binding at pH 7.4 did not induce acquired immunity to thetarget antigen in mice when it was administered to human FcRn transgenicmouse model with immune tolerance for the human IL-6 receptor; however,the antibody (Fv4-F157) produced by introducing, to the IgG1 antibodywith pH-dependent binding (Fv4-IgG1), alterations that enhanceFcRn-binding at pH 7.4 induced acquired immunity against the targetantigen in mice following administration to the mice.

However, while the aforementioned model is a model that induces acquiredimmunity against a human antigen administered from the outside, whenexamining the clinical applications of the above-described induction ofacquired immunity, it is preferable that the immunotolerance of thecompletely immunotolerant endogenous antigen is breached and whetheracquired immunity is induced is confirmed.

By using the property that human IL-6 receptor-expressing human IL-6receptor knock-in mice have immune tolerance to the endogenous humanIL-6 receptor, the aforementioned antibody was administered to the miceto assess whether acquired immunity can be induced against theendogenous human IL-6 receptor.

(4-2) Antibody Production

H54/L28-mF3 containing H54-mF3 (SEQ ID NO: 124) and L28-mCK (SEQ ID NO:125) was produced as a normal anti-human IL-6 receptor antibody withenhanced binding to mouse FcRn. Fv4-mIgG1 containing VH3-mIgG1 (SEQ IDNO: 126) and VL3-mCK (SEQ ID NO: 127), and Fv4-mIgG2a containingVH3-mIgG2a (SEQ ID NO: 128) and VL3-mCK (SEQ ID NO: 127) were producedas pH-dependent anti-human IL-6 receptor IgG antibodies. Fv4-mF3containing VH3-mF3 (SEQ ID NO: 129) and VL3-mCK (SEQ ID NO: 127) wasproduced as a pH-dependent anti-human IL-6 receptor IgG antibody withenhanced binding to mouse FcRn. Furthermore, Fv4-mFa30 containingVH3-mFa30 (SEQ ID NO: 130) and VL3-mCK (SEQ ID NO: 127) was produced asa pH-dependent anti-human IL-6 receptor IgG antibody with enhancedbinding to mouse FcRn as well as enhanced binding to mouse FcγR. Theseantibodies were prepared using the method described in Reference Example1.

(4-3) Measurement of Affinity to Mouse FcRn and Mouse FcγR

Using the values determined according to the method shown in (3-3),affinities to mouse FcRn at pH7.0 were determined for the Fc portionsmIgG1, mIgG2a, mF3, and mFa30 of the produced Fv4-mIgG1, Fv4-mIgG2a,Fv4-mF3, and Fv4-mFa30.

The extracellular domain of FcγR was prepared by the following method.First, based on the sequence information registered at NCBI, the gene ofthe extracellular domain of FcγR was synthesized by a method known tothose skilled in the art. Specifically, genes encoding the extracellulardomains of FcγR with a His-tag added to the C-terminal end of each ofthe polypeptides of NCBI Accession Number NP_034316 (Version numberNP_034316.1) for mFcγRI, NCBI Accession Number NP_034317 (Version numberNP_034317.1) for mFcγRII, NCBI Accession Number NP_034318 (Versionnumber NP_034318.2) for FcγRIII, and NCBI Accession Number NP_653142(Version number NP_653142.2) for FcγRIV were produced.

The obtained gene fragments were inserted into an animal cell expressionvector to produce expression vectors. The expression vectors wereintroduced transiently into human embryonic kidney cancer cell-derivedFreeStyle 293 cells (Invitrogen), and the culture supernatant of thetransduced cells expressing the proteins of interest was passed througha 0.22-um filter to obtain the culture supernatant. As a general rule,the extracellular domains of each of the FcγRs were purified from theobtained culture supernatant by the following four purification steps:ion exchange column chromatography in step 1 (step 1), affinity columnchromatography for the His tag (HisTrap HP) (step 2), gel filtrationcolumn chromatography (Superdex200) (step 3), and aseptic filtration(step 4). The column for ion exchange column chromatography of step 1was Q sepharose HP for purification of mFcγRI, SP Sepharose FF forpurification of mFcγRII and mFcγRIV, and SP Sepharose HP forpurification of mFcγRIII. In step 3 and subsequent purification steps,D-PBS(−) was used as the solvent, but for mFcγRIII purification,D-PBS(−) containing 0.1 M arginine was used. The absorbance at 280 nm ofthe purified solution containing the extracellular domain of FcγR wasmeasured using a spectrophotometer. From the obtained absorbance values,the concentrations of the purified extracellular domain of FcγR werecalculated using the absorption coefficient calculated by methods suchas PACE (Protein Science (1995) 4, 2411-2423).

Analysis of interaction between each of the altered antibodies and theextracellular domain of the Fcγ receptor prepared as mentioned above wascarried out using Biacore T100 (GE Healthcare), Biacore T200 (GEHealthcare), Biacore A100, and Biacore 4000. HBS-EP+(GE Healthcare) wasused for the running buffer, and the interactions were measured with ameasurement temperature at 25° C. Chips produced by immobilizing ProteinL (ACTIGEN or BioVision) by the amine coupling method to a Series Ssensor Chip CM5 (GE Healthcare) or Series S sensor Chip CM4 (GEHealthcare) were used.

After capturing of each of the altered antibodies onto these sensorchips, the extracellular domain of the Fcγ receptor diluted with therunning buffer was allowed to act on the chips to measure the bindinglevel of each of the domains to each of the antibodies, and the bindinglevels were compared. However, since the amount of the boundextracellular domain of the Fcγ receptor depends on the amount of theantibodies captured on the sensorchip, the amount of the boundextracellular domain of the Fcγ receptor was divided by the respectiveamount of captured antibody to obtain corrected values, and these valueswere compared. Furthermore, by reaction with 10 mM glycine-HCl having pHof 1.5, antibodies captured onto the sensor chips were washed, and theregenerated sensor chips were used repeatedly.

The KD values of each of the altered antibodies for the extracellulardomain of the Fcγ receptor were calculated according to the followingkinetic analysis method. Antibodies of interest were captured onto theabove mentioned sensor chips, the extracellular domain of the Fcγreceptor diluted with the running buffer was allowed to interact, and byusing the Biacore Evaluation Software to the obtained sensorgram toglobally fit the measured results using the 1:1 Langmuir binding model,the association rate constant ka (L/mol/s) and the dissociation rateconstant kd (1/s) were calculated, and from those values thedissociation constants KD (mol/L) were determined.

The affinities of mIgG1, mIgG2a, mF3, and mFa30 for mouse FcRn are shownin Table 8, and their affinity for mouse FcγR are shown in Table 9.

TABLE 8 Variant name KD (M) IgG1 Not detected mIgG2a Not detected mF31.5E−09 mFa30 3.5E−09

TABLE 9 KD (M) Variant name mFc γ RI mFc γ RII mFc γ RIII mFc γ RIV IgG1Not detected 5.7E−07 8.5E−08 Not detected mIgG2a 3.9E−09 4.8E−07 4.5E−083.2E−09 mF3 Not detected 8.7E−07 1.4E−07 Not detected mFa30 9.9E−108.1E−09 5.3E−09 1.9E−08

(4-4) Change in Plasma Concentration of the Antigen (Soluble Human IL-6Receptor) in the Antibody Administration Test

Next, Fv4-mIgG1, Fv4-mIgG2a, Fv4-mF3, Fv4-mFa30, and H54/L28-mF3 wereadministered to human IL-6 receptor knock-in mice (Reference Example25), and blood was collected from these mice over time. The collectedblood was immediately centrifuged at 15,000 rpm and 4° C. for 15 minutesto obtain the plasma. The separated plasma was stored in a freezer setat −20° C. or lower until performing the measurements. The plasmahsIL-6R concentration was measured by the same method as that describedin Example 1.

The changes in mean plasma hsIL-6R concentration for the antibodynon-administered group, and the Fv4-mIgG1, Fv4-mIgG2a, Fv4-mF3,Fv4-mFa30, and H54/L28-mF3 administration groups are shown in FIG. 26.An increase in hsIL-6R concentration was observed in the plasma of theFv4-mIgG1 and Fv4-mIgG2a administration groups. On the other hand, asignificant decrease in hsIL-6R concentration as compared to that of theantibody non-administered group was observed in the plasma of theFv4-mF3, H54/L28-mF3, and Fv4-mFa30 administration groups, where Fv4-mF3and H54/L28-mF3 have enhanced binding to mouse FcRn and Fv4-mFa30 hasenhanced binding to mouse FcRn and mouse FcgR.

(4-5) Change in Antibody Titer of the Mouse Antibody (Mouse Anti-HumanIL-6 Receptor Antibody) Against an Antigen (Soluble Human IL-6 Receptor)in Mice in an Antibody Administration Test

The anti-hsIL-6R antibody titer in mouse plasma was measured byelectrochemiluminescence. Mouse plasma samples diluted 50-fold andanti-Fv4 idiotype antibody adjusted to 30 μg/mL were mixed and reactedat room temperature for one hour. The idiotype antibody was obtained bypurifying serum from an Fv4-M73 (WO 2009/125825) immunized rabbit on anion exchange resin, and then performing affinity purification on acolumn to which Fv4-M73 has been immobilized, and subsequently adsorbingonto a human immobilized column. To the aforementioned mixed solution,50 μg/mL of a solution containing 1 μg/mL of hsIL-6R that has beenbiotinylated using an EZ-Link Sulfo-NHS-Biotin and Biotinylation Kit(Pierce) and 2 μg/mL of SULFO-anti mouse IgG (H+L) antibody (BECKMANCOULTER) that has been ruthenium-labeled using SULFO-TAG NHS Ester (MesoScale Discovery) was added, and this was mixed and allowed to reactovernight at 4° C. Under these circumstances, to prevent binding of theadministration sample included in the measurement sample with hsIL-6Rand detection of ADA directed to the administered sample, an excessamount of anti-Fv4 idiotype antibody was added to the sample in advance.Thereafter, the aforementioned reaction solution was dispensed into anMA400 PR Streptavidin Plate (Meso Scale Discovery). To each of the wellsthat were further reacted at 25° C. for one hour and then washed, Readbuffer T (×4) (Meso Scale Discovery) was dispensed, and absorbance ofthe reaction solution in each well was measured immediately using aSECTOR PR 400 reader (Meso Scale Discovery).

The changes in mouse anti-human IL-6 receptor antibody (anti-hsIL-6Rantibody) titer for each individual of the antibody non-administrationgroup, and the Fv4-mIgG1, Fv4-mIgG2a, Fv4-mF3, Fv4-mFa30, andH54/L28-mF3 administration groups are shown in FIGS. 27 to 31. Anincrease in mouse anti-human IL-6 receptor antibody (anti-hsIL-6Rantibody) titer was not observed in the individuals of theFv4-mIgG1-administered, Fv4-mIgG2a-administered, andH54/L28-mF3-administered groups, where Fv4-mIgG1 and Fv4-mIgG2a do nothave enhanced binding to mouse FcRn and H54/L28-mF3 has enhanced bindingto mouse FcRn but does not have pH-dependent binding to the human IL-6receptor. On the other hand, individuals showing an increase in mouseanti-human IL-6 receptor antibody (anti-hsIL-6R antibody) titer wereconfirmed in the Fv4-mF3-administered and Fv4-mFa30-administered groups,where Fv4-mF3 is a pH dependent binding antibody with enhanced bindingto mouse FcRn and Fv4-mFa30 is a pH-dependent binding antibody withenhanced binding to mouse FcRn and also enhanced binding to mouse FcgR.

From the above, it was shown that an antigen-binding molecule having apH-dependent target-antigen-binding activity and enhanced FcRn-bindingat pH7.4 is able to induce acquired immunity against an immunologicallytolerated endogenous target antigen. Accordingly, since such a moleculemay be able to induce acquired immunity to self cancer antigens, it isvery promising as a therapeutic agent for cancer.

Reference Example 1

The antibodies were expressed by the method described below. Humanembryonic kidney cancer cell-derived HEK293H cell line (Invitrogen) wassuspended in DMEM medium (Invitrogen) supplemented with 10% Fetal BovineSerum (Invitrogen) and plated at 10 ml per dish in dishes for adherentcells (10 cm in diameter; CORNING) at a cell density of 5×10⁵ to 6×10⁵cells/ml. The cells were cultured in a CO₂ incubator (37° C., 5% CO₂)for a whole day and night, then the medium was removed by aspiration,and 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added to the dishes.Prepared plasmids were introduced into the cells by the lipofectionmethod. The culture supernatants were collected, and centrifuged(approximately 2000 g, 5 min, room temperature) to remove cells. Theculture supernatants were further sterilized by filtering through a0.22-μm filter MILLEX(R)-GV (Millipore) to obtain culture supernatants.The expressed antibodies were purified from the obtained culturesupernatants by a method known to those skilled in the art usingrProtein A Sepharose™ Fast Flow (Amersham Biosciences). To determine theconcentration of the purified antibody, absorbance was measured at 280nm using a spectrophotometer, and antibody concentrations werecalculated from the measured values using an absorbance coefficientcalculated by the method described in Protein Science (1995) 4,2411-2423).

[Reference Example 2] Exploration of Human Germline Sequences that Bindto Calcium Ion

(2-1) Antibody that Binds to Antigen in a Calcium-Dependent Manner

Antibodies that bind to an antigen in a Ca-dependent manner(Ca-dependent antigen-binding antibodies) are those whose interactionswith antigen change with calcium concentration. A Ca-dependentantigen-binding antibody is thought to bind to an antigen throughcalcium ion. Thus, amino acids that form an epitope on the antigen sideare negatively charged amino acids that can chelate calcium ions oramino acids that can be a hydrogen-bond acceptor. These properties ofamino acids that form an epitope allows targeting of an epitope otherthan binding molecules, which are generated by introducing histidinesand bind to an antigen in a pH-dependent manner. Furthermore, as shownin FIG. 13, the use of antigen-binding molecules having calcium- andpH-dependent antigen-binding properties is thought to allow theformation of antigen-binding molecules that can individually targetvarious epitopes having broad properties. Thus, if a population ofmolecules containing a calcium-binding motif (Ca library) isconstructed, from which antigen-binding molecules are obtained,Ca-dependent antigen-binding antibodies are thought to be effectivelyobtained.

(2-2) Acquisition of Human Germline Sequences

An example of the population of molecules containing a calcium-bindingmotif is an example in which said molecules are antibodies. In otherwords, an antibody library containing a calcium-binding motif may be aCa library.

Calcium ion-binding antibodies containing human germline sequences havenot been reported. Thus, the germline sequences of antibodies havinghuman germline sequences were cloned using as a template cDNA preparedfrom Human Fetal Spleen Poly RNA (Clontech) to assess whether antibodieshaving human germline sequences bind to calcium ion. Cloned DNAfragments were inserted into animal cell expression vectors. Thenucleotide sequences of the constructed expression vectors weredetermined by a method known to those skilled in the art. The SEQ IDsare shown in Table 10. By PCR, polynucleotides encoding SEQ ID NO: 5(Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), SEQ ID NO: 8 (Vk4), andSEQ ID NO: 43 (Vk5) were linked to a polynucleotide encoding the naturalKappa chain constant region (SEQ ID NO: 44). The linked DNA fragmentswere inserted into animal cell expression vectors. Furthermore,polynucleotides encoding SEQ ID NO: 46 (Vk1), SEQ ID NO: 47 (Vk2), SEQID NO: 48 (Vk3), SEQ ID NO: 49 (Vk4), and SEQ ID NO: 45 (Vk5) werelinked by PCR to a polynucleotide encoding a polypeptide (SEQ ID NO: 11)having a deletion of two amino acids at the C terminus of IgG1. Theresulting DNA fragments were inserted into animal cell expressionvectors. The sequences of the constructed variants were confirmed by amethod known to those skilled in the art.

TABLE 10 Light SEQ ID NO of heavy SEQ ID NO of light chain germlinesequence chain variable region chain variable region Vk1 46 5 Vk2 47 6Vk3 48 7 Vk4 49 8 Vk5 45 43

(2-3) Expression and Purification of Antibodies

The constructed animal cell expression vectors inserted with the DNAfragments having the five types of human germ-line sequences wereintroduced into animal cells. Antibody expression was carried out by thefollowing method. Cells of human fetal kidney cell-derived FreeStyle293-F (Invitrogen) were suspended in the FreeStyle 293 Expression Medium(Invitrogen), and plated at a cell density of 1.33×10⁶ cells/ml (3 ml)into each well of a 6-well plate. The prepared plasmids are introducedinto cells by a lipofection method. The cells were cultured for fourdays in a CO₂ incubator (37° C., 8% CO₂, 90 rpm). From the culturesupernatants prepared as described above, antibodies were purified usingthe rProtein A Sepharose™ Fast Flow (Amersham Biosciences) by a methodknown to those skilled in the art. Absorbance at 280 nm of the purifiedantibody solutions was measured using a spectrophotometer. Antibodyconcentrations were calculated from the determined values using anextinction coefficient calculated by the PACE method (Protein Science(1995) 4: 2411-2423).

(2-4) Assessment of Antibodies Having Human Germ-Line Sequences fortheir Calcium Ion-Binding activity

The purified antibodies were assessed for their calcium ion-bindingactivity. The intermediate temperature of thermal denaturation (Tmvalue) was measured by differential scanning calorimetry (DSC) as anindicator for examining calcium ion binding to the antibody (MicroCalVP-Capillary DSC, MicroCal). The intermediate temperature of thermaldenaturation (Tm value) is an indicator of stability. It becomes higherwhen a protein is stabilized through calcium ion binding, as comparedwith the case where no calcium ion is bound (J. Biol. Chem. (2008) 283,37, 25140-25149). The binding activity of calcium ion to antibody wasevaluated by examining changes in the Tm value of the antibody dependingon the changes in the calcium ion concentration in the antibodysolution. The purified antibody was subjected to dialysis (EasySEP,TOMY) using an external solution of 20 mM Tris-HCl, 150 mM NaCl, and 2mM CaCl₂ (pH 7.4) or 20 mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl₂ (pH7.4). DSC measurement was conducted at a heating rate of 240° C./hr from20 to 115° C. using as a test substance an antibody solution prepared atabout 0.1 mg/mL with the dialysate. The intermediate temperatures ofthermal denaturation (Tm values) of the Fab domains of each antibody,calculated from the denaturation curve obtained by DSC, are shown inTable 11.

TABLE 11 Light chain Calcium ion germline concentration ΔTm (° C.)sequence 3 μM 2 mM 2 mM − 3 μM hVk1 80.32 80.78 0.46 hVk2 80.67 80.61−0.06 hVk3 81.64 81.36 −0.28 hVk4 70.74 70.74 0 hVk5 71.52 74.17 2.65

The result showed that the Tm values of the Fab domains of antibodieshaving the hVk1, hVk2, hVk3, or hVk4 sequence did not vary depending onthe calcium ion concentration in the Fab domain-containing solutions.Meanwhile, the Tm value for the antibody Fab domain having the hVk5sequence varied depending on the calcium ion concentration in the Fabdomain-containing solution. This demonstrates that the hVk5 sequencebinds to calcium ion.

(2-5) Assessment of the hVk5-2 Sequence for Calcium Binding

In addition to Vk5-2 (SEQ ID NO: 50 produced by fusing the kappa chainconstant region SEQ ID NO: 44 to SEQ ID NO: 43), Vk5-2 variant 1 (SEQ IDNO: 51) and Vk5-2 variant 2 (SEQ ID NO: 52) classified as Vk5-2 wereobtained in Reference Example 2, (2-2). These variants were assessed fortheir calcium binding activities. The DNA fragments of VK5-2, Vk5-2variant 1, and Vk5-2 variant 2 were each incorporated into expressionvectors for animal cells. The nucleotide sequences of the obtainedexpression vectors were determined by a method known to those skilled inthe art. By the method described in Reference Example 2, (2-3), theanimal cell expression vectors inserted with DNA fragments for each ofVk5-2, Vk5-2 variant 1, and Vk5-2 variant 2 were introduced into animalcells together with an animal expression vector carrying an insert toexpress CIM_H (SEQ ID NO: 45) as a heavy chain, and antibodies werepurified. The purified antibodies were assessed for their calciumion-binding activity. The purified antibodies were dialyzed (EasySEP,TOMY) against an external solution of 20 mM Tris-HCl, 150 mM NaCl, 2 mMCaCl₂ (pH 7.5), or an external solution of 20 mM Tris-HCl, 150 mM NaCl(pH 7.5) (indicated as a calcium ion concentration of 0 mM in Table 12).DSC measurement was carried out at a rate of temperature increase of240° C./hr from 20° C. to 115° C., using as the test substance, antibodysolutions prepared at a concentration of 0.1 mg/mL using the samesolution as that for dialysis. Based on the obtained DSC denaturationcurves, the intermediate temperature of thermal denaturation (Tm value)was calculated for the Fab domain of each antibody, and is shown inTable 12.

TABLE 12 Calcium ion concentration ΔTm (° C.) Light chain 0 mM 2 mM 2 mM− 0 mM Vk5-2 71.65 74.38 2.73 Vk5-2 variant 1 65.75 72.24 6.49 Vk5-2variant 2 66.46 72.24 5.78

The result showed that the Tm value for the Fab domains of antibodieshaving the sequence of Vk5-2, Vk5-2 variant 1, or Vk5-2 variant 2 varieddepending on the calcium ion concentration in solutions containingantibodies having the Fab domains. This demonstrates that antibodieshaving a sequence classified as Vk5-2 bind to calcium ion.

[Reference Example 3] Assessment of the Human Vk5 (hVk5) Sequence

(3-1) hVk5 Sequence

The only hVk5 sequence registered in Kabat's database is hVk5-2sequence. Hereinafter, hVk5 and hVk5-2 are used synonymously.WO2010/136598 discloses that the abundance ratio of the hVk5-2 sequencein the germline sequence is 0.4%. Other reports have been also made inwhich the abundance ratio of the hVk5-2 sequence in the germlinesequence is 0-0.06% (J. Mol. Biol. (2000) 296, 57-86; Proc. Natl. Acad.Sci. USA (2009) 106, 48, 20216-20221). As described above, since thehVk5-2 sequence is a sequence of low appearance frequency in thegermline sequence, it was thought to be inefficient to obtain acalcium-binding antibody from an antibody library consisting of humangermline sequences or B cells obtained by immunizing a mouse expressinghuman antibodies. Thus, it is possible to design Ca libraries containingthe sequence of human hVk5-2. Meanwhile, reported synthetic antibodylibraries (WO2010/105256 and WO2010/136598) did not contain the sequenceof hVk5. In addition, realization of the possibility is unknown becauseno report has been published on the physicochemical properties of thehVk5-2 sequence.

(3-2) Construction, Expression, and Purification of a Non-GlycosylatedForm of the hVk5-2 Sequence

The hVk5-2 sequence has a sequence for potential N glycosylation atposition 20 amino acid (Kabat's numbering). Sugar chains attached toproteins exhibit heterogeneity. Thus, it is desirable to avoid theglycosylation from the viewpoint of substance homogeneity. In thiscontext, variant hVk5-2_L65 (SEQ ID NO: 53) in which the Asn (N) residueat position 20 (Kabat's numbering) is substituted with Thr (T) wasconstructed. Amino acid substitution was carried out by a method knownto those skilled in the art using the QuikChange Site-DirectedMutagenesis Kit (Stratagene). A DNA encoding the variant hVk5-2_L65 wasinserted into an animal expression vector. The animal expression vectorinserted with the constructed DNA encoding variant hVk5-2_L65, incombination with an animal expression vector having an insert to expressCIM_H (SEQ ID NO: 45) as a heavy chain, was introduced into animal cellsby the method described in Reference Example 2. The antibody comprisinghVk5-2_L65 and CIM_H, which was expressed in animal cells introducedwith the vectors, was purified by the method described in ReferenceExample 2.

(3-3) Assessment of the Antibody Having the Non-Glycosylated hVk5-2Sequence for Physicochemical Properties

The isolated antibody having the modified sequence hVk5-2_L65 wasanalyzed by ion-exchange chromatography to test whether it is lessheterogeneous than the antibody having the original sequence hVk5-2before modification. The procedure of ion-exchange chromatography isshown in Table 13. The analysis result showed that hVk5-2_L65 modifiedat the glycosylation site was less heterogeneous than the originalsequence hVk5-2, as shown in FIG. 14.

TABLE 13 CONDITION COLUMN TOSOH TSKgel DEAE-NPR MOBILE PHASE A; 10 mMTris-HCl, 3 μM CaCl₂ (pH 8.0) B; 10 mM Tris-HCl, 500 mM NaCl, 3 μM CaCl₂(pH 8.0) GRADIENT SCHEDULE % B = 0-(5 min)-0-2%/1 min COLUMN TEMPERATURE40° C. DETECTION 280 nm INJECTION VOLUME 100 μL (5 μg)

Next, whether the less-heterogeneous hVk5-2_L65 sequence-comprisingantibody binds to calcium ion was assessed by the method described inReference Example 2. The result showed that the Tm value for the Fabdomain of the antibody having hVk5-2_L65 with altered glycosylation sitealso varied depending on the calcium ion concentration in the antibodysolutions, as shown in Table 14. Specifically, it was demonstrated thatthe Fab domain of the antibody having hVk5-2_L65 with alteredglycosylation site binds to calcium ion.

TABLE 14 CALCIUM ION CONCEN- LIGHT GLYCOSYLATED TRATION Δ Tm (° C.)CHAIN SEQUENCE 3 μM 2 mM 2 mM − 3 μM hVk5-2 YES 71.52 74.17 2.65hVk5-2_L65 NO 71.51 73.66 2.15

[Reference Example 4] Assessment of the Calcium Ion-Binding Activity ofAntibody Molecules Having CDR Sequence of the hVk5-2 Sequence

(4-1) Construction, Expression, and Purification of Modified AntibodiesHaving a CDR Sequence from the hVk5-2 Sequence

The hVk5-2_L65 sequence is a sequence with altered amino acids at aglycosylation site in the framework of human Vk5-2 sequence. Asdescribed in Reference Example 3, it was demonstrated that calcium ionbound even after alteration of the glycosylation site. Meanwhile, fromthe viewpoint of immunogenicity, it is generally desirable that theframework sequence is a germ-line sequence. Thus, the present inventorsassessed whether an antibody framework sequence could be substitutedwith the framework sequence of a non-glycosylated germline sequencewhile maintaining the calcium ion-binding activity of the antibody.

Polynucleotides encoding chemically synthesized sequences which comprisean altered framework sequence of the hVk5-2 sequence, hVk1, hVk2, hVk3,or hVk4 (CaVk1 (SEQ ID NO: 54), CaVk2 (SEQ ID NO: 55), CaVk3 (SEQ ID NO:56), or CaVk4 (SEQ ID NO: 57), respectively) were linked by PCR to apolynucleotide encoding the constant region (SEQ ID NO: 44) of thenatural Kappa chain. The linked DNA fragments were inserted into animalcell expression vectors. Sequences of the constructed variants wereconfirmed by a method known to those skilled in the art. Each plasmidconstructed as described above was introduced into animal cells incombination with a plasmid inserted with a polynucleotide encoding CIM_H(SEQ ID NO: 45) by the method described in Reference Example 2. Theexpressed antibody molecules of interest were purified from culturefluid of the animal cells introduced with the plasmids.

(4-2) Assessment of Altered Antibodies Having the CDR Sequence of thehVk5-2 Sequence for their Calcium Ion-Binding Activity

Whether calcium ion binds to altered antibodies having the CDR sequenceof the hVk5-2 sequence and the framework sequences of germline sequencesother than hVk5-2 (hVk1, hVk2, hVk3, and hVk4) was assessed by themethod described in Reference Example 2. The assessment result is shownin Table 15. The Tm value of the Fab domain of each altered antibody wasrevealed to vary depending on the calcium ion concentration in theantibody solutions. This demonstrates that antibodies having a frameworksequence other than the framework sequences of the hVk5-2 sequence alsobind to calcium ion.

TABLE 15 GERMLINE (LIGHT CHAIN CALCIUM FRAMEWORK ION CONCENTRATION Δ Tm(° C.) SEQUENCE) 3 μM 2 mM 2 mM − 3 μM hVk1 77.51 79.79 2.28 hVk2 78.4680.37 1.91 hVk3 77.27 79.54 2.27 hVk4 80.35 81.38 1.03 hVk5-2 71.5274.17 2.65

The thermal denaturation temperature (Tm value), as an indicator ofthermal stability, of the Fab domain of each antibody altered to havethe CDR sequence of the hVk5-2 sequence and the framework sequence of agerm-line sequence other than the hVk5-2 sequence (hVk1, hVk2, hVk3, orhVk4) was demonstrated to be greater than that of the Fab domain of theoriginal antibody having the hVk5-2 sequence. This result shows thatantibodies having the CDR sequence of the hVk5-2 sequence and theframework sequence of hVk1, hVk2, hVk3, or hVk4 not only have calciumion-binding activity but also are excellent molecules from the viewpointof thermal stability.

[Reference Example 5] Identification of the Calcium Ion-Binding Site inHuman Germline hVk5-2 Sequence

(5-1) Design of Mutation Site in the CDR Sequence of the hVk5-2 Sequence

As described in Reference Example 4, antibodies having the light chainresulting from introduction of the CDR domain of the hVk5-2 sequenceinto the framework sequence of a different germline sequence were alsodemonstrated to bind to calcium ion. This result suggests that in hVk5-2a calcium ion-binding site is localized within its CDR. Amino acids thatbind to calcium ion, i.e., chelate calcium ion, include negativelycharged amino acids and amino acids that can be a hydrogen bondacceptor. Thus, it was tested whether antibodies having a mutant hVk5-2sequence with a substitution of an Ala (A) residue for an Asp (D) or Glu(E) residue in the CDR sequence of the hVk5-2 sequence bind to calciumion.

(5-2) Construction of Variant hVk5-2 Sequences with Ala Substitution,and Expression and Purification of Antibodies

Antibody molecules were prepared to comprise a light chain withsubstitution of an Ala residue for Asp and/or Glu residue in the CDRsequence of the hVk5-2 sequence. As described in Reference Example 3,non-glycosylated variant hVk5-2_L65 exhibited calcium ion binding andwas assumed to be equivalent to the hVk5-2 sequence in terms of calciumion binding. In this Reference Example, amino acid substitutions wereintroduced into hVk5-2_L65 as a template sequence. Constructed variantsare shown in Table 16. Amino acid substitutions were carried out bymethods known to those skilled in the art such as using the QuikChangeSite-Directed Mutagenesis Kit (Stratagene), PCR, or the In fusionAdvantage PCR Cloning Kit (TAKARA) to construct expression vectors foraltered light chains having an amino acid substitution.

TABLE 16 Light chain Altered position variant name (Kabat numbering) SEQID NO: hVk5-2_L65 Wild type 53 hVk5-2_L66 30 58 hVk5-2_L67 31 59hVk5-2_L68 32 60 hVk5-2_L69 50 61 hVk5-2_L70 30, 32 62 hVk5-2_L71 30, 5063 hVk5-2_L72 30, 32, 50 64 hVk5-2_L73 92 65

Nucleotide sequences of the constructed expression vectors wereconfirmed by a method known to those skilled in the art. The expressionvectors constructed for the altered light chains were transientlyintroduced, in combination with an expression vector for the heavy chainCIM_H (SEQ ID NO: 45), into cells of the human fetal kidney cell-derivedHEK293H line (Invitrogen) or FreeStyle293 (Invitrogen) to expressantibodies. From the obtained culture supernatants, antibodies werepurified using the rProtein A Sepharose™ Fast Flow (GE Healthcare) by amethod known to those skilled in the art. Absorbance at 280 nm of thepurified antibody solutions was measured using a spectrophotometer.Antibody concentrations were calculated from the determined values usingan extinction coefficient calculated by the PACE method (Protein Science(1995) 4: 2411-2423).

(5-3) Assessment of the Calcium Ion-Binding Activity of AntibodiesHaving an Ala Substitution in the hVk5-2 Sequence

Whether the obtained purified antibodies bind to calcium ion was testedby the method described in Reference Example 2. The result is shown inTable 17. Some antibodies having substitution of an Asp or Glu residuein the CDR sequence of the hVk5-2 sequence with an Ala residue whichcannot be involved in calcium ion binding or chelation were revealed tohave an Fab domain whose Tm did not vary by the calcium ionconcentration in the antibody solutions. The substitution sites at whichAla substitution did not alter the Tm (positions 32 and 92 (Kabat'snumbering)) were demonstrated to be greatly important for the calciumion-antibody binding.

TABLE 17 LIGHT CALCIUM ION CHAIN ALTERED CONCEN- VARIANT POSITION(Kabat's TRATION Δ Tm (° C.) NAME NUMBERING) 0 μM 2 mM 2 mM − 0 μMhVk5-2_L65 WILDTYPE 71.71 73.69 1.98 hVk5-2_L66 30 71.65 72.83 1.18hVk5-2_L67 31 71.52 73.30 1.78 hVk5-2_L68 32 73.25 74.03 0.78 hVk5-2_L6950 72.00 73.97 1.97 hVk5-2_L70 30, 32 73.42 73.60 0.18 hVk5-2_L71 30, 5071.84 72.57 0.73 hVk5-2_L72 30, 32, 50 75.04 75.17 0.13 hVk5-2_L73 9275.23 75.04 −0.19

[Reference Example 6] Assessment of the Calcium Ion-Binding Activity ofAntibodies Having hVk1 Sequence with Calcium Ion-Binding Motif

(6-1) Construction of an hVk1 Sequence with Calcium Ion-Binding Motif,and Expression and Purification of Antibodies

The result described in Reference Example 4 on the calcium-bindingactivity of the Ala substitute demonstrates that Asp or Glu residues inthe CDR sequence of the hVk5-2 sequence were important for calciumbinding. Thus, the present inventors assessed whether an antibody canbind to calcium ion when the residues at positions 30, 31, 32, 50, and92 (Kabat's numbering) alone were introduced into a different germlinevariable region sequence. Specifically, variant LfVk1_Ca (SEQ ID NO: 66)was constructed by substituting the residues at positions 30, 31, 32,50, and 92 (Kabat's numbering) in the hVk5-2 sequence for the residuesat positions 30, 31, 32, 50, and 92 (Kabat's numbering) in the hVk1sequence (a human germline sequence). Specifically, it was testedwhether antibodies having an hVk1 sequence introduced with only 5residues from the hVk5-2 sequence can bind to calcium. The variants wereproduced by the same method as described in Reference Example 5. Theresulting light chain variant LfVk1_Ca and LfVk1 having the light-chainhVk1 sequence (SEQ ID NO: 67) were co-expressed with the heavy chainCIM_H (SEQ ID NO: 45). Antibodies were expressed and purified by thesame method as described in Reference Example 4.

(6-2) Assessment of the Calcium Ion-Binding Activity of AntibodiesHaving a Human hVk1 Sequence with Calcium Ion-Binding Motif

Whether the purified antibody prepared as described above binds tocalcium ion was assessed by the method described in Reference Example 2.The result is shown in Table 18. The Tm value of the Fab domain of theantibody having LfVk1 with an hVk1 sequence did not vary depending onthe calcium concentration in the antibody solutions. Meanwhile, Tm ofthe antibody having the LfVk1_Ca sequence was shifted by 1° C. or moreupon change in the calcium concentration in the antibody solutions.Thus, it was shown that the antibody having LfVk1 Ca binds to calcium.The result described above demonstrates that the entire CDR sequence ofhVk5-2 is not required, while the residues introduced for constructionof the LfVk1_Ca sequence alone are sufficient for calcium ion binding.

TABLE 18 CALCIUM LIGHT CHAIN ION CONCENTRATION Δ Tm (° C.) VARIANT 3 μM2 mM 2 mM − 3 μM LfVk1 83.18 83.81 0.63 LfVk1_Ca 79.83 82.24 2.41

[Reference Example 7] Design of a Population of Antibody Molecules (CaLibrary) with a Calcium Ion-Binding Motif Introduced into the VariableRegion to Effectively Obtain Binding Antibodies that Bind to Antigen ina Ca Concentration-Dependent Manner

Preferred calcium-binding motifs include, for example, the hVk5-2sequence and the CDR sequence, as well as residues at positions 30, 31,32, 50, and 92 (Kabat numbering). Other calcium-binding motifs includethe EF-hand motif possessed by calcium-binding proteins (e.g.,calmodulin) and C-type lectin (e.g., ASGPR).

The Ca library is composed of heavy chain variable regions and lightchain variable regions. Human antibody sequences were used for the heavychain variable regions and a calcium-binding motif was introduced to thelight chain variable regions. The hVk1 sequence was selected as atemplate sequence of the light chain variable region to which acalcium-binding motif is inserted. The antibody containing the LfVk1_Casequence, which has the CDR sequence of hVk5-2, one of thecalcium-binding motifs, introduced into the hVk1 sequence, was shown tobind to a calcium ion as shown in Reference Example 5. Multiple aminoacids were allowed to appear in the template sequence to diversifyantigen-binding molecules that constitute the library. Positions exposedon the surface of a variable region which is likely to interact with theantigen were selected as those where multiple amino acids are allowed toappear. Specifically, positions 30, 31, 32, 34, 50, 53, 91, 92, 93, 94,and 96 (Kabat numbering) were selected as flexible residues.

The type and appearance frequency of amino acid residues that weresubsequently allowed to appear were determined. The appearance frequencyof amino acids in the flexible residues of the hVk1 and hVk3 sequencesregistered in the Kabat database (KABAT, E. A. ET AL.: ‘Sequences ofproteins of immunological interest’, vol. 91, 1991, NIH PUBLICATION) wasanalyzed. Based on the analysis results, the type of amino acids thatwere allowed to appear in the Ca library were selected from those withhigher appearance frequency at each position. At this time, amino acidswhose appearance frequency was determined to be low based on theanalysis results were also selected to avoid the bias of amino acidproperties. The appearance frequency of the selected amino acids wasdetermined in reference to the analysis results of the Kabat database.

A Ca library containing a calcium-binding motif with emphasis on thesequence diversity as to contain multiple amino acids at each residueother than the motif were designed as a Ca library in consideration ofthe amino acids and appearance frequency set as described above. Thedetailed designs of the Ca library are shown in Tables 1 and 2 (with thepositions in each table representing the Kabat numbering). When position92 based on the Kabat numbering is Asn (N), the frequency of appearanceof amino acids shown in Tables 1 and 2 may be Leu (L) rather than Ser(S) at position 94.

(Reference Example 8) Ca Library Production

A library of antibody heavy chain variable region genes was amplified bythe PCR method using poly A RNA prepared from human PBMC or commerciallyavailable human poly A RNA as template. Regarding the antibody lightchain variable region portions, as shown in Reference Example 7,antibody variable region light chain portions which increase thefrequency of appearance of antibodies maintaining a calcium-bindingmotif and allows binding to antigens in acalcium-concentration-dependent manner were designed. In addition, foramino acid residues among the flexible residues other than those with acalcium-binding motif introduced, a library of antibody light chainvariable regions with evenly distributed amino acids of high appearancefrequency in natural human antibody sequences was designed withreference to the information of amino acid appearance frequency innatural human antibodies (KABAT, E. A. ET AL.: ‘Sequences of proteins ofimmunological interest’, vol. 91, 1991, NIH PUBLICATION). A combinationof the gene libraries of antibody heavy-chain and light-chain variableregions generated as described above, was inserted into a phagemidvector to construct a human antibody phage display library that presentsFab domains consisting of human antibody sequences (Methods Mol Biol.(2002) 178, 87-100).

Sequences of the antibody gene portions isolated from E. coli carryingthe antibody gene library were confirmed. The amino acid distribution ofthe sequences of the obtained 290 clones and the designed amino aciddistribution are shown in FIG. 15.

(Reference Example 9) Assessment of Calcium Ion-Binding Activity ofMolecules Included in the Ca Library (9-1) Calcium Ion-Binding Activityof Molecules Included in the Ca Library

As shown in Reference Example 3, the hVk5-2 sequence shown to bind tocalcium ions has low frequency of appearance in the germ line sequences;therefore, trying to obtain calcium-binding antibodies from antibodylibraries composed of human germline sequences or from B cells obtainedby immunization of human antibody-expressing mice was consideredinefficient. Accordingly, a Ca library was constructed in ReferenceExample 8. The constructed Ca library was assessed for the presence ofclones showing calcium binding.

(9-2) Expression and Purification of Antibodies

Clones of the Ca library were introduced into animal cell expressionplasmids. Antibodies were expressed using the method described below.Cells of human fetal kidney cell-derived FreeStyle 293-F line(Invitrogen) were suspended in FreeStyle 293 Expression Medium(Invitrogen), and plated at a cell density of 1.33×10⁶ cells/ml (3 ml)to each well of a 6-well plate. The prepared plasmids were introducedinto the cells by a lipofection method. The cells were cultured in a CO₂incubator (37° C., 8% CO₂, 90 rpm) for four days. By a method known tothose skilled in the art, antibodies were purified using rProtein ASepharose™ Fast Flow (Amersham Biosciences) from culture supernatantsobtained as described above. The absorbance of solutions of purifiedantibodies was measured at 280 nm using a spectrophotometer. Antibodyconcentrations were calculated from the measured values by using theabsorption coefficient determined by PACE method (Protein Science (1995)4, 2411-2423).

(9-3) Assessment of Calcium Ion-Binding Property of the ObtainedAntibodies

Whether the purified antibodies obtained as described above bind tocalcium ions was assessed by the method described in Example 6. Theresults are shown in Table 19. The Tm of the Fab domains of multipleantibodies included in the Ca library changed depending on the calciumion concentration, and this showed that the library includescalcium-ion-binding molecules.

TABLE 19 Calcium ion SEQ ID NO: concentration ΔTm (° C.) Antibody Heavychain Light chain 3 μM 2 mM 2 mM − 3 μM Ca_B01 68 79 70.88 71.45 0.57Ca_E01 69 80 84.31 84.95 0.64 Ca_H01 70 81 77.87 79.49 1.62 Ca_D02 71 8278.94 81.1 2.16 Ca_E02 72 83 81.41 83.18 1.77 Ca_H02 73 84 72.84 75.132.29 Ca_D03 74 85 87.39 86.78 −0.61 Ca_C01 75 86 74.74 74.92 0.18 Ca_G0176 87 65.21 65.87 0.66 Ca_A03 77 88 80.64 81.89 1.25 Ca_B03 78 89 93.0293.75 0.73

[Reference Example 10] Preparation of Antibodies that Bind to IL-6Receptor in a Ca-Dependent Manner

(10-1) Preparation of Antibody Fragments that Bind to the Antigen in aCa-Dependent Manner from Library by Bead Panning

Primary selection from the constructed library of antibodies that bindto IL-6 receptor in a Ca-dependent manner was carried out by enrichingantibody fragments that have antigen (IL-6 receptor)-binding activity.

Phages were produced from E. coli carrying the constructed phagemid forphage display. To precipitate the phages produced by E. coli, 2.5 MNaCl/10% PEG was added to the E. coli culture fluid. The phage fractionwas diluted with TBS to prepare a phage library solution. Then, BSA andCaCl₂ were added the phage library solution at final concentrations of4% and 1.2 mM calcium ion, respectively. The panning method used was aconventional panning method using antigen-immobilized magnetic beads (J.Immunol. Methods. (2008) 332(1-2): 2-9; J. Immunol. Methods. (2001)247(1-2): 191-203; Biotechnol. Prog. (2002) 18(2): 212-20; Mol. CellProteomics (2003) 2(2): 61-9). The magnetic beads used wereNeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) andStreptavidin-coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution. Thus, the solution was contacted withthe antigen at room temperature for 60 minutes. Magnetic beads blockedwith BSA were added, and the antigen-phage complex was allowed to bindto the magnetic beads at room temperature for 15 minutes. The beads werewashed three times with 1 mL of 1.2 mM CaCl₂/TBST (TBST containing 1.2mM CaCl₂), and then twice with 1 ml of 1.2 mM CaCl₂/TBS (TBS containing1.2 mM CaCl₂). Thereafter, 0.5 ml of 1 mg/ml trypsin was added to thebeads. After 15 minutes of dispersion at room temperature, the beadswere immediately separated using a magnetic stand to collect a phagesuspension. The prepared phage suspension was added to 10 ml of E. coliof stain ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7). TheE. coli was incubated with gentle stirring at 37° C. for one hour toinfect the phages. The infected E. coli was seeded in a plate (225mm×225 mm). Then, phages were collected from the culture fluid of theseeded E. coli to prepare a phage library solution.

In the second round panning, phages were enriched using theantigen-binding activity or the Ca-dependent binding activity as anindicator.

Specifically, when the enrichment was carried out using theantigen-binding ability as an indicator, 40 pmol of biotin-labeledantigen was added to the prepared phage library solution to allow thecontact of the phage library solution with the antigen at roomtemperature for 60 minutes. BSA-blocked magnetic beads were added andallowed to bind to antigen/phage complexes at room temperature for 15minutes. The beads were washed three times with 1 ml of 1.2 mMCaCl₂/TBST and then twice with 1.2 mM CaCl₂/TBS. Then, the beads addedwith 0.5 ml of 1 mg/ml trypsin were suspended at room temperature for 15minutes. Then immediately, the beads were separated using a magneticstand to collect a phage solution. To eliminate the ability from phagesdisplaying on Fab to infect E. coli, the pIII protein (helperphage-derived pIII protein) of phages displaying no Fab was cleaved byadding 5 μl of 100 mg/ml trypsin to the collected phage solution. Thecollected phage solution was added to 10 mL of the E. coli strain ER2738in a logarithmic growth phase (OD600 of 0.4-0.7). The E. coli wascultured with gentle stirring at 37° C. for 1 hour to allow the phagesto infect the E. coli. The infected E. coli was inoculated into a 225mm×225 mm plate. Subsequently, the phages were collected from theculture fluid of the E. coli after inoculation to collect a phagelibrary solution.

When the enrichment was carried out using the Ca-dependent bindingability as an indicator, 40 pmol of biotin-labeled antigen was added tothe prepared phage library solution to allow the contact of the phagelibrary solution with the antigen at room temperature for 60 minutes.BSA-blocked magnetic beads were added and allowed to bind toantigen/phage complexes at room temperature for 15 minutes. The beadswere washed with 1 ml of 1.2 mM CaCl₂/TBST and with 1.2 mM CaCl₂/TBS.Then, the beads added with 0.1 ml of 2 mM EDTA/TBS (TBS containing 2 mMEDTA) were suspended at room temperature. Then immediately, the beadswere separated using a magnetic stand to collect a phage solution. Toeliminate the ability from phages displaying on Fab to infect E. coli,the pIII protein (helper phage-derived pIII protein) of phagesdisplaying no Fab was cleaved by adding 5 μl of 100 mg/ml trypsin to thecollected phage solution. The collected phage solution was added to 10mL of the E. coli strain ER2738 in a logarithmic growth phase (OD600 of0.4-0.7). The E. coli was cultured with gentle stirring at 37° C. for 1hour to allow the phages to infect the E. coli. The infected E. coli wasinoculated into a 225 mm×225 mm plate. Subsequently, the phages werecollected from the culture fluid of the E. coli after inoculation tocollect a phage library solution.

(10-2) Assessment by Phage ELISA

Culture supernatants containing phages were collected from singlecolonies of E. coli obtained by the method described above according toa conventional method (Methods Mol. Biol. (2002) 178, 133-145).

BSA and CaCl₂ were added to the phage-containing culture supernatants.The supernatants were subjected to ELISA by the following procedure. AStreptaWell 96-well microtiter plate (Roche) was coated overnight with100 μl of PBS containing the biotin-labeled antigen. The antigen wasremoved by washing each well of the plate with PBST. Then, the wellswere blocked with 250 μl of 4% BSA-TBS for one hour or more. Afterremoval of 4% BSA-TBS, the prepared culture supernatants were added tothe each well. The plate was incubated at 37° C. for one hour so thatthe antibody-displaying phages were allowed to bind to the antigen oneach well. After each well was washed with 1.2 mM CaCl₂/TBST, 1.2 mMCaCl₂/TBS or 1 mM EDTA/TBS was added. The plate was left for incubationat 37° C. for 30 minutes. After washing with 1.2 mM CaCl₂/TBST, anHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS at an ionized calcium concentration of 1.2 mM was added to eachwell, and the plate was incubated for one hour. After washing with 1.2mM CaCl₂/TBST, the TMB single solution (ZYMED) was added to each well.The chromogenic reaction in the solution of each well was stopped byadding sulfuric acid. Then, the developed color was assessed bymeasuring absorbance at 450 nm.

To clones subjected to the above phage ELISA, the base sequence of agene amplified with specific primers was analyzed. The result ofsequence analysis was shown in Table 20 below.

TABLE 20 Library Ca library Ca library Enrichment index Antigen-bindingDependent antigen- ability binding ability Number of panning 2 2 Numberof examined clones 85 86 ELISA-positive 77 75 Types of ELISA-positiveclone 74 72 sequences Types of Ca-dependent 13 47 binding clonesequences

(10-3) Expression and Purification of Antibodies

Clones that were judged to have a Ca-dependent antigen-binding activitybased on the result of phage ELISA were inserted into animal cellexpression plasmids. Antibody expression was carried out by thefollowing method. Cells of human fetal kidney cell-derived FreeStyle293-F (Invitrogen) were suspended in the FreeStyle 293 Expression Medium(Invitrogen), and plated at a cell density of 1.33×10⁶ cells/ml (3 ml)into each well of a 6-well plate. The prepared plasmids were introducedinto cells by a lipofection method. The cells were cultured for fourdays in a CO₂ incubator (37° C., 8% CO₂, 90 rpm). From the obtainedculture supernatants, antibodies were purified using the rProtein ASepharose™ Fast Flow (Amersham Biosciences) by a method known to thoseskilled in the art. Absorbance at 280 nm of the purified antibodysolutions was measured using a spectrophotometer. Antibodyconcentrations were calculated from the determined values using anextinction coefficient calculated by the PACE method (Protein Science(1995) 4: 2411-2423).

(10-4) Assessment of Ca-Dependent Binding Ability to the Human IL-6Receptor for the Obtained Antibodies

To determine whether the antibodies obtained in Reference Example 9,i.e. 6RC1IgG_010 (heavy chain SEQ ID NO: 90, light chain SEQ ID NO: 91),6RC1IgG_012 (heavy chain SEQ ID NO: 92, light chain SEQ ID NO: 93), and6RC1IgG_019 (heavy chain SEQ ID NO: 94, light chain SEQ ID NO: 95), haveCa-dependent binding activity to the human IL-6 receptor, analyses ofinteraction between these antibodies and the human IL-6 receptor werecarried out using Biacore T100 (GE Healthcare). Tocilizumab (heavy chainSEQ ID NO: 96, light chain SEQ ID NO: 97) was used as the controlantibody which does not have Ca-dependent binding activity to the humanIL-6 receptor. Interaction analyses were performed in a solution with acalcium ion concentration of 1.2 mM for the high calcium ionconcentration condition or 3 μM for the low calcium ion concentrationcondition. A suitable amount of protein A/G (Invitrogen) was immobilizedonto a Sensor chip CM5 (GE Healthcare) by the amine coupling method, andthen antibodies of interest were captured onto the chips. Two types ofbuffers, 20 mM ACES, 150 mM NaCl, 0.05% (w/v) Tween 20, 1.2 mM CaCl₂ (pH7.4) or 20 mM ACES, 150 mM NaCl, 0.05% (w/v) Tween 20, 3 μM CaCl₂ (pH7.4), were used as the running buffer. The respective buffers were alsoused for dilution of the human IL-6 receptor. All measurements weretaken at 37° C.

When performing interaction analyses on antigen-antibody reactions usingthe tocilizumab antibody, which is the control antibody, the 6RC1IgG_010antibody, the 6RC1IgG_012 antibody, and the 6RC1IgG_019 antibody, thediluted IL-6 receptor solution and the running buffer, which is theblank, were injected at a flow rate of 5 μL/min for three minutes toallow the IL-6 receptor to interact with the tocilizumab antibody, the6RC1IgG_010 antibody, the 6RC1IgG_012 antibody, and the 6RC1IgG_019antibody captured onto the sensor chip. Then, 10 mM glycine-HCl (pH 1.5)was injected at a flow rate of 30 μL/min for 30 seconds to regeneratethe sensor chip.

Sensorgrams at high calcium ion concentration obtained by this methodare shown in FIG. 16.

Sensorgrams for the tocilizumab antibody, the 6RC1IgG_010 antibody, the6RC1IgG_012 antibody, and the 6RC1IgG_019 antibody under low calcium ionconcentration conditions were also obtained by a similar method. Thesensorgrams obtained at low calcium ion concentration are shown in FIG.17.

From the above-mentioned results, ability of the 6RC1IgG_010 antibody,the 6RC1IgG_012 antibody, and the 6RC1IgG_019 antibody to bind to theIL6 receptor was observed to be greatly reduced by setting the calciumion concentration in the buffer to 3 μM from 1.2 mM.

[Reference Example 11] Acquisition of Antibodies that Bind to IL-6Receptor in Ca-Dependent Manner from a Human Antibody Library UsingPhage Display Technology (11-1) Preparation of a Phage Display Libraryfor Naive Human Antibodies

A phage display library for human antibodies, consisting of multiplephages presenting the Fab domains of mutually different human antibodysequences, was constructed according to a method known to those skilledin the art using a poly A RNA prepared from human PBMC, and commercialhuman poly A RNA as a template.

(11-2) Acquisition of Antibody Fragments that Bind to Antigen inCa-Dependent Manner from the Library by Bead Panning

The constructed phage display library for naive human antibodies wassubjected to initial selection through concentration of only antibodyfragments having an antigen (IL-6 receptor)-binding ability orconcentration of antibody fragments using a Ca concentration-dependentantigen (IL-6 receptor)-binding ability as an indicator. Concentrationof antibody fragments using a Ca concentration-dependent antigen (IL-6receptor)-binding ability as an indicator were conducted through elutionof the phage library phages bound to IL-6 receptor in the presence of Caions with EDTA that chelates the Ca ions Biotinylated IL-6 receptor wasused as an antigen.

Phages were produced from Escherichia coli carrying the constructedphage display phagemid. A phage library solution was obtained bydiluting with TBS a phage population precipitated by adding 2.5 MNaCl/10% PEG to the E. coli culture solution in which the phages wereproduced. Subsequently, BSA and CaCl₂ were added to the phage librarysolution at a final concentration of 4% BSA and 1.2 mM of calcium ionconcentration. A common panning method using an antigen immobilized onmagnetic beads was referred to as a panning method (J. Immunol. Methods.(2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203;Biotechnol. Prog. (2002) 18(2) 212-20; Mol. Cell Proteomics (2003) 2(2), 61-9). NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280Streptavidin) were used as magnetic beads.

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution to allow the contact of said phagelibrary solution with the antigen for 60 minutes at room temperature.Magnetic beads, blocked with BSA, were added to be bound toantigen-phage complexes for 15 minutes at room temperature. The beadswere washed once with 1 mL of 1.2 mM CaCl₂/TBS (TBS containing 1.2 mMCaCl₂). Subsequently, a phage solution was collected by a generalelution method to concentrate an antibody fragment having an IL-6receptor-binding ability, or by elution from beads suspended in 2 mMEDTA/TBS (TBS containing 2 mM EDTA) to concentrate an antibody fragmentusing an IL-6 receptor-binding ability in a Ca concentration-dependentmanner as an indicator. The collected phage solution was added to 10 mLof the E. coli strain TG1 in a logarithmic growth phase (OD600 of0.4-0.7). The E. coli was cultured with gentle stirring at 37° C. for 1hour to allow the phages to infect the E. coli. The infected E. coli wasinoculated into a 225 mm×225 mm plate. Subsequently, the phages werecollected from the culture fluid of the E. coli after inoculation toprepare a phage library solution.

In the second and subsequent panning, the phages were concentrated usingthe Ca-dependent binding ability as an indicator. Specifically, 40 pmolof the biotin-labeled antigen was added to the prepared phage librarysolution to allow the contact of the phage library with the antigen for60 minutes at room temperature. Magnetic beads, blocked with BSA, wereadded to be bound to antigen-phage complexes for 15 minutes at roomtemperature. The beads were washed with 1 mL of 1.2 mM CaCl₂/TBST and1.2 mM CaCl₂/TBS. Subsequently, the beads, to which 0.1 mL of 2 mMEDTA/TBS was added, were suspended at room temperature. Immediatelyafter that, the beads were separated using a magnetic stand to collect aphage solution. The collected phage solution was added to 10 mL of theE. coli strain TG1 in a logarithmic growth phase (OD600 of 0.4-0.7). TheE. coli was cultured with gentle stirring at 37° C. for 1 hour to allowthe phages to infect the E. coli. The infected E. coli was inoculatedinto a 225 mm×225 mm plate. Subsequently, the phages were collected fromthe culture fluid of the E. coli after inoculation to collect a phagelibrary solution. The panning using the Ca-dependent binding ability asan indicator was repeated several times.

(11-3) Examination by Phage ELISA

A phage-containing culture supernatant was collected according to aroutine method (Methods Mol. Biol. (2002) 178, 133-145) from a singlecolony of E. coli, obtained as described above.

A culture supernatant containing phages, to which BSA and CaCl₂ wereadded at a final concentration of 4% BSA and 1.2 mM of calcium ionconcentration was subjected to ELISA as described below. A StreptaWell96 microtiter plate (Roche) was coated overnight with 100 μL of PBScontaining the biotin-labeled antigen. Each well of said plate waswashed with PBST to remove the antigen, and then the wells were blockedwith 250 μL of 4% BSA-TBS for 1 hour or longer. Said plate with theprepared culture supernatant added to each well, from which the 4%BSA-TBS was removed, was allowed to stand undisturbed at 37° C. for 1hour, allowing the binding of phage-presenting antibody to the antigenpresent in each well. To each well washed with 1.2 mM CaCl₂/TBST, 1.2 mMCaCl₂/TBS or 1 mM EDTA/TBS was added. The plate was allowed to standundisturbed for 30 minutes at 37° C. for incubation. After washing with1.2 mM CaCl₂/TBST, an HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS at a final concentration of 4% BSAand 1.2 mM of ionized calcium concentration was added to each well, andthe plate was incubated for 1 hour. After washing with 1.2 mMCaCl₂/TBST, the chromogenic reaction of the solution in each well with aTMB single solution (ZYMED) added was stopped by adding sulfuric acid.Subsequently, said color was measured by measuring absorbance at 450 nm.

As a result of the above phage ELISA, the base sequence of a geneamplified with specific primers and an antibody fragment identified ashaving a Ca-dependent antigen-binding ability as a template wasanalyzed.

(11-4) Antibody Expression and Purification

As a result of the above phage ELISA, a clone identified as having aCa-dependent antigen-binding ability was introduced into an expressionplasmid for animal cells. Antibodies were expressed as described below.FreeStyle 293-F strain (Invitrogen) derived from human fetal kidneycells was suspended in FreeStyle 293 Expression Medium (Invitrogen),followed by inoculation of 3 mL into each well of a 6-well plate at acell density of 1.33×10⁶ cell/mL. The prepared plasmid was introducedinto the cells by lipofection. The cells were cultured for 4 days in aCO₂ incubator (37° C., 8% CO₂, 90 rpm). Antibodies were purified fromthe culture supernatant obtained above by a method known in the artusing rProtein A Sepharose (trade mark) Fast Flow (AmershamBiosciences). Absorbance of the purified antibody solution was measuredat 280 nm using a spectrophotometer. Antibody concentration wascalculated from the measurements obtained using an extinctioncoefficient calculated by the PACE method (Protein Science (1995) 4,2411-2423).

[Reference Example 12] Examination of Ca-Dependent Binding Ability ofthe Obtained Antibodies to Human IL-6 Receptor

To examine whether or not the binding activities of antibodies6RL#9-IgG1 [heavy chain (a sequence in which a constant region derivedfrom IgG1 is linked to SEQ ID NO: 98) and light chain (SEQ ID NO: 99)]and FH4-IgG1 [heavy chain (SEQ ID NO: 100) and light chain (SEQ ID NO:101)], obtained in Reference Example 11, to human IL-6 receptor areCa-dependent, the kinetic analysis of the antigen-antibody reactions ofthese antibodies with human IL-6 receptor was conducted using BiacoreT100 (GE Healthcare). H54/L28-IgG1 [heavy chain: SEQ ID NO: 102; andlight chain: SEQ ID NO: 103], described in WO2009/125825, was used as acontrol antibody that has no Ca-dependent binding activity to human IL-6receptor. The kinetic analysis of the antigen-antibody reactions wasconducted in solutions with 2 mM and 3 μM calcium ion concentrations,set as high and low calcium ion concentration conditions, respectively.The antibody of interest was captured on Sensor chip CM4 (GE Healthcare)on which an appropriate amount of protein A (Invitrogen) was immobilizedby an amine coupling method. Two buffers [10 mM ACES, 150 mM NaCl, 0.05%(w/v) Tween 20, and 2 mM CaCl₂ (pH 7.4) or 10 mM ACES, 150 mM NaCl,0.05% (w/v) Tween 20, and 3 μmol/L CaCl₂ (pH 7.4)] were used as runningbuffers. These buffers were used for diluting human IL-6 receptor. Allthe measurements were conducted at 37° C.

In the kinetic analysis of antigen-antibody reaction using H54L28-IgG1antibody, the H54L28-IgG1 antibody captured on the sensor chip wasallowed to interact with IL-6 receptor by injecting a diluent of IL-6receptor and running buffer (blank) at a flow rate of 20 μL/min for 3minutes. Subsequently, after the dissociation of IL-6 receptor wasobserved using running buffer at a flow rate of 20 μL/min for 10minutes, the sensor chip was regenerated by injecting 10 mM glycine-HCl(pH 1.5) at a flow rate 30 μL/min for 30 seconds. Kinetics parameters,binding constant (ka) (1/Ms) and dissociation rate constant (kd) (1/s),were calculated from the sensorgrams obtained in the measurement. Thesevalues were used to calculate the dissociation constant (KD) (M) of theH54L28-IgG1 antibody for human IL-6 receptor. Each parameter wascalculated using the Biacore T100 Evaluation Software (GE Healthcare).

In the kinetic analysis of antigen-antibody reaction using FH4-IgG1 and6RL#9-IgG1 antibodies, the FH4-IgG1 or 6RL#9-IgG1 antibody captured onthe sensor chip was allowed to interact with IL-6 receptor by injectinga diluent of IL-6 receptor and running buffer (blank) at a flow rate of5 μL/min for 15 minutes. Subsequently, the sensor chip was regeneratedby injecting 10 mM glycine-HCl (pH 1.5) at a flow rate 30 μL/min for 30seconds. Dissociation constants (KD) (M) were calculated from thesensorgrams obtained in the measurement, using a steady-state affinitymodel. Each parameter was calculated using the Biacore T100 EvaluationSoftware (GE Healthcare).

The dissociation constants (KD) between each antibody and IL-6 receptorin the presence of 2 mM CaCl₂, determined by the above method, are shownin Table 21.

TABLE 21 ANTIBODY H54/L28-IgG1 FH4-IgG1 6RL#9-IgG1 kD (M) 1.9E−9 5.9E−72.6E−7

The KD value of the H54/L28-IgG1 antibody under the condition of 3 μM Caconcentration can be calculated in the same manner as in the presence of2 mM Ca concentration. Under the condition of 3 μM Ca concentration,FH4-IgG1 and 6RL#9-IgG1 antibodies were barely observed to be bound toIL-6 receptor, thus the calculation of KD values by the method describedabove is difficult. However, the KD values of these antibodies under thecondition of 3 μM Ca concentration can be estimated using Equation 1(Biacore T100 Software Handbook, BR-1006-48, AE 01/2007) below.

Req=C×Rmax/(KD+C)+RI  [Equation 1]

The meaning of each parameter in the aforementioned [Equation 1] is asfollows:Req (RU): Steady state binding levelsRmax (RU): Analyte binding capacity of the surfaceRI (RU): Bulk refractive index contribution in the sampleC (M) Analyte concentrationKD (M): Equilibrium dissociation constant

The approximate results of dissociation constant KD values for theantibodies and IL-6 receptor at a Ca concentration of 3 μmol/L,estimated using the above-described [Equation 1], are shown in Table 22.In Table 22, the Req, Rmax, RI, and C values are estimated based on theassay result.

TABLE 22 ANTIBODY H54/L28-IgG1 FH4-IgG1 6RL#9-IgG1 Req (RU) 5 10 Rmax(RU) 39  72 RI (RU) 0  0 C (M)   5E−06   5E−06 KD (M) 2.2E−9 3.4E−053.1E−05

Based on the findings described above, it was predicted that the KDbetween IL-6 receptor and FH4-IgG1 antibody or 6RL#9-IgG1 antibody wasincreased by about 60 or 120 times (the affinity was reduced by 60 or120 times or more) when the concentration of CaCl₂ in the buffer wasdecreased from 2 mM to 3 μM.

Table 23 summarizes the KD values at CaCl₂ concentrations of 2 mM and 3μM and the Ca dependency for the three types of antibodies H54/L28-IgG1,FH4-IgG1, and 6RL#9-IgG1.

TABLE 23 H54/ ANTIBODY L28-IgG1 FH4-IgG1 6RL#9-IgG1 KD (M) 1.9E−9 5.9E−72.6E−7 (2 mM CaCl₂) KD (M) 2.2E−9 3.4E−5 OR MORE 3.1E−5 OR MORE (3 μMCaCl₂) Ca ABOUT ABOUT 60 TIMES ABOUT 120 DEPENDENCY THE OR MORE TIMES ORSAME MORE

No difference in the binding of the H54/L28-IgG1 antibody to IL-6receptor due to the difference in Ca concentration was observed. On theother hand, the binding of FH4-IgG1 and 6RL#9-IgG1 antibodies to IL-6receptor was observed to be significantly attenuated under the conditionof the low Ca concentration (Table 23).

[Reference Example 13] Examination of Calcium Ion Binding to theAntibody Obtained

Subsequently, the intermediate temperature of thermal denaturation (Tmvalue) was measured by differential scanning calorimetry (DSC) as anindicator for examining calcium ion binding to the antibody (MicroCalVP-Capillary DSC, MicroCal). The intermediate temperature of thermaldenaturation (Tm value) is an indicator of stability. The intermediatetemperature of thermal denaturation (Tm value) becomes higher when aprotein is stabilized through calcium ion binding, as compared with nocalcium ion binding (J. Biol. Chem. (2008) 283, 37, 25140-25149). Thebinding activity of calcium ion to antibody was examined by examiningchanges in the Tm value of the antibody depending on the changes in thecalcium ion concentration of the antibody solution. The purifiedantibody was subjected to dialysis (EasySEP, TOMY) using an externalsolution of 20 mM Tris-HCl, 150 mM NaCl, and 2 mM CaCl₂ (pH 7.4), or 20mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl₂ (pH 7.4). DSC measurement wasconducted at a heating rate of 240° C./hr from 20 to 115° C. using anantibody solution prepared at about 0.1 mg/mL with the dialysate as atest substance. The intermediate temperatures of thermal denaturation(Tm values) of the Fab domains of each antibody, calculated based on thedenaturation curve obtained by DSC, are shown in Table 24.

TABLE 24 CALCIUM ION CONCENTRATION ΔTm (° C.) ANTIBODY 3 μM 2 mM 2 mM −3 μM H54/L28-IgG1 92.87 92.87 0.00 FH4-IgG1 74.71 78.97 4.26 6RL#9-IgG177.77 78.98 1.21

From the results shown in Table 24, it is indicated that the Tm valuesof the Fab of the FH4-IgG1 and 6RL#9-IgG1 antibodies, which show acalcium-dependent binding ability, varied with changes in the calciumion concentration, while the Tm values of the Fab of the H54/L28-IgG1antibody which shows no calcium-dependent binding ability do not varywith changes in the calcium ion concentration. The variation in the Tmvalues of the Fab of the FH4-IgG1 and 6RL#9-IgG1 antibodies demonstratesthat calcium ions bound to these antibodies to stabilize the Fabportions. The above results show that calcium ions bound to the FH4-IgG1and 6RL#9-IgG1 antibodies, while no calcium ion bound to theH54/L28-IgG1 antibody.

[Reference Example 14] Identification of Calcium Ion-Binding Site inAntibody 6RL#9 by X-Ray Crystallography (14-1) X-Ray Crystallography

As described in Reference Example 13, the measurements of thermaldenaturation temperature Tm suggested that antibody 6RL#9 binds tocalcium ion. However, it was unpredictable which portion of antibody6RL#9 binds to calcium ion. Then, by using the technique of X-raycrystallography, residues of antibody 6RL#9 that interact with calciumion were identified.

(14-2) Expression and Purification of Antibody 6RL#9

Antibody 6RL#9 was expressed and purified for X-ray crystallography.Specifically, animal expression plasmids constructed to be capable ofexpressing the heavy chain (a sequence in which a constant regionderived from IgG1 is linked to SEQ ID NO: 98) and light chain (SEQ IDNO: 99) of antibody 6RL#9 were introduced transiently into animal cells.The constructed plasmids were introduced by the lipofection method intocells of human fetal kidney cell-derived FreeStyle 293-F (Invitrogen)suspended in 800 ml of the FreeStyle 293 Expression Medium (Invitrogen)(final cell density: 1×10⁶ cells/mL). The plasmid-introduced cells werecultured in a CO₂ incubator (37° C., 8% CO₂, 90 rpm) for five days. Fromthe culture supernatant obtained as described above, antibodies werepurified by a method known to those skilled in the art using therProtein A Sepharose® Fast Flow (Amersham Biosciences). Absorbance at280 nm of purified antibody solutions was measured using aspectrophotometer. Antibody concentrations were calculated from themeasured values using an extinction coefficient calculated by the PACEmethod (Protein Science (1995) 4, 2411-2423).

(14-3) Purification of Antibody 6RL#9 Fab Fragment

Antibody 6RL#9 was concentrated to 21 mg/ml using an ultrafilter with amolecular weight cutoff of 10,000 MWCO. A 5 mg/mL antibody sample (2.5mL) was prepared by diluting the antibody solution using 4 mML-cysteine/5 mM EDTA/20 mM sodium phosphate buffer (pH 6.5). 0.125 mg ofpapain (Roche Applied Science) was added to the sample. After stirring,the sample was incubated at 35° C. for two hours. After incubation, atablet of Protease Inhibitor Cocktail Mini, EDTA-free (Roche AppliedScience) was dissolved in 10 ml of 25 mM MES buffer (pH 6) and added tothe sample. The sample was incubated on ice to stop the papainproteolytic reaction. Then, the sample was loaded onto a 1-mlcation-exchange column HiTrap SP HP (GE Healthcare) equilibrated with 25mM MES buffer (pH 6), downstream of which a 1-ml HiTrap MabSelect SureProtein A column (GE Healthcare) was connected in tandem. A purifiedfraction of the Fab fragment of antibody 6RL#9 was obtained byperforming elution with a linear NaCl concentration gradient up to 300mM in the above-described buffer. Then, the resulting purified fractionwas concentrated to about 0.8 ml using a 5000 MWCO ultrafilter. Theconcentrate was loaded onto a gel filtration column Superdex 200 10/300GL (GE Healthcare) equilibrated with 100 mM HEPES buffer (pH 8)containing 50 mM NaCl. The purified Fab fragment of antibody 6RL#9 forcrystallization was eluted from the column using the same buffer. Allthe column treatments described above were carried out at a lowtemperature of 6 to 7.5° C.

(14-4) Crystallization of the Antibody 6RL#9 Fab Fragment in thePresence of Ca

Seed crystals of the 6RL#9 Fab fragment were prepared in advance undergeneral conditions. Then, the purified Fab fragment of antibody 6RL#9 in5 mM CaCl₂ was concentrated to 12 mg/ml with a 5000 MWCO ultrafilter.Next, the sample concentrated as described above was crystallized by thehanging drop vapor diffusion method using 100 mM HEPES buffer (pH 7.5)containing 20% to 29% PEG4000 as a reservoir solution. Theabove-described seed crystals were crushed in 100 mM HEPES buffer (pH7.5) containing 29% PEG4000 and 5 mM CaCl₂, and serially diluted to 100to 10,000 folds. Then, 0.2 μL of diluted solutions were combined with amixture of 0.8 μl of the reservoir solution and 0.8 μl of theconcentrated sample to prepare crystallization drops on a glass coverslide. The crystal drops were allowed to stand at 20° C. for two tothree days to prepare thin plate-like crystals. X-ray diffraction datawere collected using the crystals.

(14-5) Crystallization of the Antibody 6RL#9 Fab Fragment in the Absenceof Ca

The purified Fab fragment of antibody 6RL#9 was concentrated to 15 mg/mlusing a 5000 MWCO ultrafilter. Then, the sample concentrated asdescribed above was crystallized by the hanging drop vapor diffusionmethod using 100 mM HEPES buffer (pH 7.5) containing 18% to 25% PEG4000as a reservoir solution. Crystals of the antibody 6RL#9 Fab fragmentobtained in the presence of Ca were crushed in 100 mM HEPES buffer (pH7.5) containing 25% PEG4000, and serially diluted to 100 to 10,000folds. Then, 0.2 μL of diluted solutions were combined with a mixture of0.8 μl of the reservoir solution and 0.8 μl of the concentrated sampleto prepare crystallization drops on a glass cover slide. The crystaldrops were allowed to stand at 20° C. for two to three days to preparethin plate-like crystals. X-ray diffraction data were collected usingthe crystals.

(14-6) X-Ray Crystallographic Measurement of Fab Fragment Crystal fromAntibody 6RL#9 in the Presence of Ca

Crystals of the Fab fragment of antibody 6RL#9 prepared in the presenceof Ca were soaked in 100 mM HEPES buffer (pH 7.5) solution containing35% PEG4000 and 5 mM CaCl₂. By removing the exterior solution from thesurface of a single crystal with a micro-nylon-loop pin, the singlecrystal was frozen in liquid nitrogen. X-ray diffraction data of thefrozen crystal was collected from beam line BL-17A of the Photon Factoryin the High Energy Accelerator Research Organization. The frozen crystalwas maintained in the frozen state during the measurement by constantlyplacing it in a stream of nitrogen gas at −178° C. A total of 180diffraction images were collected using the CCD detector Quantum315r(ADSC) attached to the beam line while rotating the crystal in 1°intervals. Lattice constant determination, diffraction spot indexing,and diffraction data analysis were performed using programs Xia2 (CCP4Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 SoftwareSuite). Finally, diffraction intensity data up to 2.2 angstromresolution was obtained. The crystal belongs to space group P212121 withlattice constant a=45.47 angstrom, b=79.86 angstrom, c=116.25 angstrom,α=90°, β=90°, and γ=90°.

(14-7) X-Ray Crystallographic Measurement of the Fab Fragment Crystalfrom Antibody 6RL#9 in the Absence of Ca

Crystals of the Fab fragment of antibody 6RL#9 prepared in the absenceof Ca were soaked in 100 mM HEPES buffer (pH 7.5) solution containing35% PEG4000. By removing the exterior solution from the surface of asingle crystal with a micro-nylon-loop pin, the single crystal wasfrozen in liquid nitrogen. X-ray diffraction data of the frozen crystalwas collected from beam line BL-5A of the Photon Factory in the HighEnergy Accelerator Research Organization. The frozen crystal wasmaintained in the frozen state during the measurement by constantlyplacing it in a stream of nitrogen gas at −178° C. A total of 180diffraction images were collected using the CCD detector Quantum210r(ADSC) attached to the beam line while rotating the crystal in 1°intervals. Lattice constant determination, diffraction spot indexing,and diffraction data analysis were performed using programs Xia2 (CCP4Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 SoftwareSuite). Finally, diffraction intensity data up to 2.3 angstromresolution was obtained. The crystal belongs to space group P212121 withlattice constant a=45.40 angstrom, b=79.63 angstrom, c=116.07 angstrom,α=90°, β=90°, γ=90°, and thus is structurally identical to the crystalprepared in the presence of Ca.

(14-8) X-Ray Crystallographic Measurement of the Fab Fragment Crystalfrom Antibody 6RL#9 in the Presence of Ca

The crystal structure of the antibody 6RL#9 Fab fragment in the presenceof Ca was determined by a molecular replacement method using the Phaserprogram (CCP4 Software Suite). The number of molecules in anasymmetrical unit was estimated to be one from the size of crystallattice and molecular weight of the antibody 6RL#9 Fab fragment. Basedon the primary sequence homology, a portion of amino acid positions 112to 220 from A chain and a portion of amino acid positions 116 to 218from B chain in the conformational coordinate of PDB code 1ZA6 were usedas model molecules for analyzing the CL and CH1 regions. Then, a portionof amino acid positions 1 to 115 from B chain in the conformationalcoordinate of PDB code 1ZA6 was used as a model molecule for analyzingthe VH region. Finally, a portion of amino acid positions 3 to 147 ofthe light chain in the conformational coordinate of PDB code 2A9M wasused as a model molecule for analyzing the VL region. Based on thisorder, an initial structure model for the antibody 6RL#9 Fab fragmentwas obtained by determining from translation and rotation functions thepositions and orientations of the model molecules for analysis in thecrystal lattice. The crystallographic reliability factor R for thereflection data at 25 to 3.0 angstrom resolution was 46.9% and Free Rwas 48.6% after rigid body refinement where the VH, VL, CH1, and CLdomains were each allowed to deviate from the initial structure model.Then, model refinement was achieved by repeating structural refinementusing program Refmac5 (CCP4 Software Suite) followed by model revisionperformed using program Coot (Paul Emsley) with reference to the Fo-Fcand 2Fo-F electron density maps where the coefficients Fo-Fc and 2Fo-Fcwere calculated using experimentally determined structural factor Fo,structural factor Fc calculated based on the model, and the phases. Thefinal refinement was carried out using program Refmac5 (CCP4 SoftwareSuite) based on the Fo-Fc and 2Fo-F electron density maps by addingwater molecule and Ca ion into the model. With 21,020 reflection data at25 to 2.2 angstrom resolution, eventually the crystallographicreliability factor R became 20.0% and free R became 27.9% for the modelconsisting of 3440 atoms.

(14-9) Measurement of X-Ray Diffraction Data of the Fab Fragment Crystalfrom Antibody 6RL#9 in the Absence of Ca

The crystal structure of the antibody 6RL#9 Fab fragment in the absenceof Ca was determined based on the structure of the crystal prepared inthe presence of Ca. Water and Ca ion molecules were omitted from theconformational coordinate of the crystal of the antibody 6RL#9 Fabfragment prepared in the presence of Ca. The crystallographicreliability factor R for the data of reflection at 25 to 3.0 angstromresolution was 30.3% and Free R was 31.7% after the rigid bodyrefinement where the VH, VL, CH1, and CL domains were each allowed todeviate. Then, model refinement was achieved by repeating structuralrefinement using program Refmac5 (CCP4 Software Suite) followed by modelrevision performed using program Coot (Paul Emsley) with reference tothe Fo-Fc and 2Fo-Fc electron density maps where the coefficients Fo-Fcand 2Fo-Fc were calculated using experimentally determined structuralfactor Fo, structural factor Fc calculated based on the model, and thephases. The final refinement was carried out using program Refmac5 (CCP4Software Suite) based on the Fo-Fc and 2Fo-F electron density maps byadding water molecule and Ca ion into the model. With 18,357 reflectiondata at 25 to 2.3 angstrom resolution, eventually the crystallographicreliability factor R became 20.9% and free R became 27.7% for the modelconsisting of 3351 atoms.

(14-10) Comparison of X-Ray Crystallographic Diffraction Data of the FabFragments of Antibody 6RL#9 Between in the Presence and Absence of Ca

When the crystallographic structures of the Fab fragments of antibody6RL#9 are compared between in the presence and absence of Ca,significant changes are seen in the heavy chain CDR3. The structure ofthe heavy chain CDR3 of the antibody 6RL#9 Fab fragment determined byX-ray crystallography is shown in FIG. 18. Specifically, a calcium ionresided at the center of the heavy chain CDR3 loop region of theantibody 6RL#9 Fab fragment prepared in the presence of Ca. The calciumion was assumed to interact with positions 95, 96, and 100a (Kabat'snumbering) of the heavy chain CDR3. It was believed that the heavy chainCDR3 loop which is important for the antigen binding was stabilized bycalcium binding in the presence of Ca, and became an optimum structurefor antigen binding. There is no report demonstrating that calcium bindsto the antibody heavy chain CDR3. Thus, the calcium-bound structure ofthe antibody heavy chain CDR3 is a novel structure.

The calcium-binding motif that was found to exist in the heavy chainCDR3 from the structure of the Fab fragment of the 6RL#9 antibody mayalso be a new component in the design of a Ca library such as thosedescribed in Reference Example 7. That is, while the calcium-bindingmotif was introduced into the light chain variable region in ReferenceExample 7, one may consider, for example, a library containing the 6RL#9antibody heavy chain CDR3 and flexible residues in other CDRs includingthe light chain.

[Reference Example 15] Preparation of Antibodies that Bind to IL-6 in aCa-Dependent Manner from a Human Antibody Library Using Phage DisplayTechniques (15-1) Construction of a Phage Display Library of Naïve HumanAntibodies

A human antibody phage display library containing multiple phages thatdisplay various human antibody Fab domain sequences was constructed by amethod known to those skilled in the art using, as a template, polyA RNAprepared from human PBMC, commercially available human polyA RNA, andsuch.

(15-2) Preparation of Antibody Fragments that Bind to the Antigen in aCa-Dependent Manner from Library by Bead Panning

Primary selection from the constructed phage display library of naïvehuman antibodies was carried out by enriching antibody fragments thathave antigen (IL-6)-binding activity. The antigen used wasbiotin-labeled IL-6.

Phages were produced from E. coli carrying the constructed phagemid forphage display. To precipitate the phages produced by E. coli, 2.5 MNaCl/10% PEG was added to the E. coli culture fluid. The phage fractionwas diluted with TBS to prepare a phage library solution. Then, BSA andCaCl₂ were added the phage library solution at final concentrations of4% and 1.2 mM calcium ion concentration, respectively. The panningmethod used was a conventional panning method using antigen-immobilizedmagnetic beads (J. Immunol. Methods. (2008) 332(1-2): 2-9; J. Immunol.Methods. (2001) 247(1-2): 191-203; Biotechnol. Prog. (2002) 18(2):212-20; Mol. Cell Proteomics (2003) 2(2): 61-9). The magnetic beads usedwere NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated)and Streptavidin-coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of the biotin-labeled antigen was added to theprepared phage library solution. Thus, the solution was contacted withthe antigen at room temperature for 60 minutes. Magnetic beads blockedwith BSA were added, and the antigen-phage complex was allowed to bindto the magnetic beads at room temperature for 15 minutes. The beads werewashed three times with 1.2 mM CaCl₂/TBST (TBST containing 1.2 mMCaCl₂), and then twice with 1 ml of 1.2 mM CaCl₂/TBS (TBS containing 1.2mM CaCl₂). Thereafter, 0.5 ml of 1 mg/ml trypsin was added to the beads.After 15 minutes of dispersion at room temperature, the beads wereimmediately separated using a magnetic stand to collect a phagesuspension. The prepared phage suspension was added to 10 ml of E. coliof stain TG1 at the logarithmic growth phase (OD600=0.4 to 0.7). The E.coli was incubated with gentle stirring at 37° C. for one hour to infectthe phages. The infected E. coli was seeded in a plate (225 mm×225 mm).Then, phages were collected from the culture fluid of the seeded E. colito prepare a phage library solution.

In the second round and subsequent panning, phages were enriched usingthe Ca-dependent binding activity as an indicator. Specifically, 40 pmolof the biotin-labeled antigen was added to the prepared phage librarysolution. Thus, the phage library was contacted with the antigen at roomtemperature for 60 minutes. Magnetic beads blocked with BSA were added,and the antigen-phage complex was allowed to bind to the magnetic beadsat room temperature for 15 minutes. The beads were washed with 1 ml of1.2 mM CaCl₂/TBST and 1.2 mM CaCl₂/TBS. Next, 0.1 ml of 2 mM EDTA/TBSwas added to the beads. After dispersion at room temperature, the beadswere immediately separated using a magnetic stand to collect a phagesuspension. The pIII protein (helper phage-derived protein pIII) wascleaved from phages that did not display Fab by adding 5 μl of 100 mg/mltrypsin to the collected phage suspension to eliminate the ability ofphages displaying no Fab to infect E. coli. Phages collected from thetrypsinized liquid phage stock was added to 10 ml of E. coli cells ofthe TG1 strain at the logarithmic growth phase (OD600=0.4 to 0.7). TheE. coli was incubated while gently stirring at 37° C. for one hour toinfect phage. The infected E. coli was seeded in a plate (225 mm×225mm). Then, phages were collected from the culture fluid of the seeded E.coli to prepare a liquid stock of phage library. Panning was performedthree times using the Ca-dependent binding activity as an indicator.

(15-3) Assessment by Phage ELISA

Culture supernatants containing phages were collected from singlecolonies of E. coli obtained by the method described above according toa conventional method (Methods Mol. Biol. (2002) 178, 133-145). BSA andCaCl₂ were added at final concentrations of 4% and 1.2 mM calcium ionconcentration, respectively, to the phage-containing culturesupernatants.

The supernatants were subjected to ELISA by the following procedure. AStreptaWell 96-well microtiter plate (Roche) was coated overnight with100 μl of PBS containing the biotin-labeled antigen. The antigen wasremoved by washing each well of the plate with PBST. Then, the wellswere blocked with 250 μl of 4% BSA-TBS for one hour or more. Afterremoval of 4% BSA-TBS, the prepared culture supernatants were added tothe each well. The plate was incubated at 37° C. for one hour so thatthe antibody-displaying phages were allowed to bind to the antigen oneach well. After each well was washed with 1.2 mM CaCl₂/TBST, 1.2 mMCaCl₂/TBS or 1 mM EDTA/TBS was added. The plate was left for incubationat 37° C. for 30 minutes. After washing with 1.2 mM CaCl₂/TBST, anHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS containing BSA and calcium ion at final concentrations of 4%and 1.2 mM calcium ion concentration was added to each well, and theplate was incubated for one hour. After washing with 1.2 mM CaCl₂/TBST,the TMB single solution (ZYMED) was added to each well. The chromogenicreaction in the solution of each well was stopped by adding sulfuricacid. Then, the developed color was assessed by measuring absorbance at450 nm.

From the 96 clones isolated, antibody 6KC4-1#85 having Ca-dependentIL-6-binding activity was obtained by phage ELISA. Using antibodyfragments that were predicted to have a Ca-dependent antigen-bindingactivity based on the result of the phage ELISA described above as atemplate, genes were amplified with specific primers and their sequenceswere analyzed. The heavy-chain and light-chain variable region sequencesof antibody 6KC4-1#85 are shown in SEQ ID NOs: 10 and 104, respectively.The polynucleotide encoding the heavy-chain variable region of antibody6KC4-1#85 (SEQ ID NO: 10) was linked to a polynucleotide encoding anIgG1-derived sequence by PCR method. The resulting DNA fragment wasinserted into an animal cell expression vector to construct anexpression vector for the heavy chain of SEQ ID NO: 105. Apolynucleotide encoding the light-chain variable region of antibody6KC4-1#85 (SEQ ID NO: 104) was linked to a polynucleotide encoding theconstant region of the natural Kappa chain (SEQ ID NO: 44) by PCR. Thelinked DNA fragment was inserted into an animal cell expression vector.Sequences of the constructed variants were confirmed by a method knownto those skilled in the art. Sequences of the constructed variants wereconfirmed by a method known to those skilled in the art.

(15-4) Expression and Purification of Antibodies

Clone 6KC4-1#85 that was predicted to have a Ca-dependentantigen-binding activity based on the result of phage ELISA was insertedinto animal cell expression plasmids. Antibody expression was carriedout by the following method. Cells of human fetal kidney cell-derivedFreeStyle 293-F (Invitrogen) were suspended in the FreeStyle 293Expression Medium (Invitrogen), and plated at a cell density of 1.33×10⁶cells/ml (3 ml) into each well of a 6-well plate. The prepared plasmidswere introduced into cells by a lipofection method. The cells arecultured for four days in a CO₂ incubator (37° C., 8% CO₂, 90 rpm). Fromthe culture supernatants, antibodies were purified using the rProtein ASepharose™ Fast Flow (Amersham Biosciences) by a method known to thoseskilled in the art. Absorbance at 280 nm of the purified antibodysolutions was measured using a spectrophotometer. Antibodyconcentrations were calculated from the determined values using anextinction coefficient calculated by the PACE method (Protein Science(1995) 4: 2411-2423).

[Reference Example 16] Assessment of Antibody 6KC4-1#85 for Calcium IonBinding

Calcium-dependent antigen-binding antibody 6KC4-1#85 which was isolatedfrom a human antibody library was assessed for its calcium binding.Whether the measured Tm value varies depending on the ionized calciumconcentration condition was assessed by the method described inReference Example 2.

Tm values for the Fab domain of antibody 6KC4-1#85 are shown in Table25. As shown in Table 25, the Tm value of the 6KC4-1#85 antibody Fabdomain varied depending on the calcium ion concentration. Thisdemonstrates that antibody 6KC4-1#85 binds to calcium.

TABLE 25 CALCIUM ION CONCENTRATION ΔTm (° C.) ANTIBODY 3 μM 2 mM 2 mM −3 μM 6KC4-1#85 71.49 75.39 3.9

[Reference Example 17] Identification of Calcium Ion-Binding Site inAntibody 6KC4-1#85

As demonstrated in Reference Example 16, antibody 6KC4-1#85 binds tocalcium ion. However, 6KC4-1#85 does not have a calcium-binding motifsuch as the hVk5-2 sequence which was revealed from assessment to have acalcium-binding motif. Then, whether calcium ion binds to either or bothof the heavy chain and the light chain of antibody 6KC4-1#85 wasconfirmed by assessing the calcium ion binding of altered antibodiesresulting from exchanging the heavy chain and light chain of 6KC4-1#85respectively with those of an anti-glypican 3 antibody (heavy chainsequence GC_H (SEQ ID NO: 106), light chain sequence GC_L (SEQ ID NO:107)) which does not bind calcium ion. The Tm values of alteredantibodies measured according to the method described in ReferenceExample 2 are shown in Table 26. The result suggests that the heavychain of antibody 6KC4-1#85 binds to calcium, because the Tm values ofthe altered antibody having the heavy chain of antibody 6KC4-1#85changed depending on calcium ion concentration.

TABLE 26 CALCIUM ION LIGHT CONCENTRATION ΔTm (° C.) HEAVY CHAIN CHAIN 3μM 2 mM 2 mM − 3 μM 6KC4-1#85 6KC4-1#85 71.46 75.18 3.72 6KC4-1#85 GC_L78.87 80.01 1.14 GC_H 6KC4-1#85 75.69 75.94 0.25 GC_H GC_L 79.94 80.010.07

Thus, to further identify residues responsible for the calcium ionbinding of antibody 6KC4-1#85, altered heavy chains (6_H1-11 (SEQ ID NO:108), 6_H1-12 (SEQ ID NO: 109), 6_H1-13 (SEQ ID NO: 110), 6_H1-14 (SEQID NO: 111), 6_H1-15 (SEQ ID NO: 112)) or altered light chains (6_L1-5(SEQ ID NO: 113) and 6_L1-6 (SEQ ID NO: 114)) were constructed bysubstituting an Asp (D) residue in the CDR of antibody 6KC4-1#85 with anAla (A) residue which does not participate in the binding or chelationof calcium ion. By the method described in Reference Example 15, alteredantibodies were purified from the culture supernatants of animal cellsintroduced with expression vectors carrying the altered antibody genes.The purified altered antibodies were assessed for their calcium bindingaccording to the method described in Reference Example 2. Themeasurement result is shown in Table 27. As shown in Table 27,substitution of an Ala residue for the residue at position 95 or 101(Kabat numbering) in the heavy chain CDR3 of antibody 6KC4-1#85 resultedin loss of the calcium-binding activity of antibody 6KC4-1#85. Thissuggests that these residues are responsible for calcium binding. Thecalcium-binding motif located at the base of the CDR3 loop in the heavychain of antibody 6KC4-1#85, which was found based on the calciumbinding capacity of the antibody altered from antibody 6KC4-1#85, can bea new factor for designing Ca libraries as described in ReferenceExample 7. In Reference Example 7, calcium-binding motifs wereintroduced into the light chain variable region. Meanwhile, suchlibraries include, for example, those containing the heavy chain CDR3from antibody 6KC4-1#85 and flexible residues in the CDRs other than theheavy chain CDR3 but including the light chain CDRs.

TABLE 27 CALCIUM ION CONCENTRATION Δ Tm (° C.) HEAVY CHAIN LIGHT CHAINALTERED RESIDUE 3 μM 2 mM 2 mM − 3 μM 6KC4-1#85 6KC4-1#85 WILD-TYPE71.49 75.39 3.9 6H1-11 6KC4-1#85 H CHAIN 71.73 75.56 3.83 POSITION 61(Kabat NUMBERING) 6H1-12 6KC4-1#85 H CHAIN 72.9 73.43 0.53 POSITION 95(Kabat NUMBERING) 6H1-13 6KC4-1#85 H CHAIN 70.94 76.25 5.31 POSITION100a (Kabat NUMBERING) 6H1-14 6KC4-1#85 H CHAIN 73.95 75.14 1.19POSITION 100g (Kabat NUMBERING) 6H1-15 6KC4-1#85 H CHAIN 65.37 66.250.87 POSITION 101 (Kabat NUMBERING) 6KC4-1#85 6L1-5 L CHAIN 71.92 76.084.16 POSITION 50 (Kabat NUMBERING) 6KC4-1#85 6L1-6 L CHAIN 72.13 78.746.61 POSITION 92 (Kabat NUMBERING)

[Reference Example 18] Examination of Effects of Ca-Dependent BindingAntibody on Plasma Retention of Antigen Using Normal Mice (18-1) In VivoTest Using Normal Mice

To a normal mouse (C57BL/6J mouse, Charles River Japan), hsIL-6R(soluble human IL-6 receptor prepared in Reference Example 21) alone wasadministered, or hsIL-6R and anti-human IL-6 receptor antibody wereadministered simultaneously to examine the kinetics of the hsIL-6R andanti-human IL-6 receptor antibody in vivo. A single dose (10 mL/kg) ofthe hsIL-6R solution (5 μg/mL) or a mixture of hsIL-6R and anti-humanIL-6 receptor antibody was administered into the caudal vein. The aboveH54/L28-IgG1, 6RL#9-IgG1, and FH4-IgG1 were used as anti-human IL-6receptor antibodies.

The hsIL-6R concentration in all the mixtures is 5 μg/mL. Theconcentrations of anti-human IL-6 receptor antibody vary with theantibodies: 0.1 mg/mL for H54/L28-IgG1 and 10 mg/mL for 6RL#9-IgG1 andFH4-IgG1. At this time, it is thought that most of the hsIL-6Rs bind tothe antibody because the anti-human IL-6 receptor antibody againsthsIL-6R exists in a sufficient or excessive amount. Blood samples werecollected at 15 minutes, 7 hours and 1, 2, 4, 7, 14, 21, and 28 daysafter the administration. The blood samples obtained were immediatelycentrifuged for 15 minutes at 4° C. and 12,000 rpm to separate plasma.The separated plasma was stored in a freezer set to −20° C. or loweruntil the time of measurement.

(18-2) Determination of Plasma Anti-Human IL-6 Receptor AntibodyConcentration in Normal Mice by ELISA

The plasma concentration of anti-human IL-6 receptor antibody in a mousewas determined by ELISA. First, Anti-Human IgG (γ-chain specific)F(ab′)2 Fragment of Antibody (SIGMA) was dispensed into a Nunc-ImmunoPlate, MaxiSoup (Nalge Nunc International), and was allowed to standundisturbed overnight at 4° C. to prepare an anti-human IgG-solid phaseplate. Calibration curve samples at a plasma concentration of 0.64,0.32, 0.16, 0.08, 0.04, 0.02, or 0.01 μg/mL, and mouse plasmameasurement samples diluted by 100-fold or above were each dispensedinto the anti-human IgG-solid phase plate, followed by incubation for 1hour at 25° C. Subsequently, the plate was allowed to react with abiotinylated anti-human IL-6 R antibody (R&D) for 1 hour at 25° C.,followed by reaction with Streptavidin-PolyHRP80 (StereospecificDetection Technologies) for 0.5 hours at 25° C. The chromogenic reactionwas conducted using TMB One Component HRP Microwell Substrate (BioFXLaboratories) as a substrate. After the chromogenic reaction was stoppedby adding 1N-sulfuric acid (Showa Chemical), absorbance at 450 nm of thecolor solution was measured using a microplate reader. The plasmaconcentration in the mouse was calculated from the absorbance of thecalibration curve using the SOFTmax PRO analysis software (MolecularDevices). Changes in the plasma concentrations of antibodies,H54/L28-IgG1, 6RL#9-IgG1, and FH4-IgG1, in the normal mice afterintravenous administration, measured as described above, are shown inFIG. 42.

(18-3) Determination of Plasma hsIL-6R Concentration by anElectrochemiluminescence Method

The plasma concentration of hsIL-6R in a mouse was determined by anelectrochemiluminescence method. An hsIL-6R calibration curve sampleprepared at 2,000, 1,000, 500, 250, 125, 62.5, or 31.25 pg/mL, and amouse plasma measurement sample diluted by 50-fold or above, were mixedwith a monoclonal anti-human IL-6R antibody (R&D) ruthenated withSULFO-TAG NHS Ester (Meso Scale Discovery), a biotinylated anti-humanIL-6 R antibody (R&D), and tocilizumab (heavy chain SEQ ID NO: 96, lightchain SEQ ID NO: 97), followed by overnight reaction at 4° C. At thattime, the assay buffer contained 10 mM EDTA to reduce the free Caconcentration in the sample and dissociate almost all the hsIL-6Rs inthe sample from 6RL#9-IgG1 or FH4-IgG1 to be bound to the addedtocilizumab. Subsequently, said reaction liquid was dispensed into anMA400 PR Streptavidin Plate (Meso Scale Discovery). In addition, afterwashing each well of the plate that was allowed to react for 1 hour at25° C., Read Buffer T (×4) (Meso Scale Discovery) was dispensed intoeach well. Immediately, the reaction liquid was subjected to measurementusing a SECTOR PR 400 reader (Meso Scale Discovery). The concentrationof hsIL-6R was calculated from the response of the calibration curveusing the SOFTmax PRO analysis software (Molecular Devices). Changes inthe plasma concentration of hsIL-6R in the normal mouse afterintravenous administration, determined as described above, are shown inFIG. 20.

As a result, the disappearance of hsIL-6R was very rapid when hsIL-6Rwas administered alone, while the disappearance of hsIL-6R wassignificantly delayed when hsIL-6R was administered simultaneously withH54/L28-IgG1, a conventional antibody having no Ca-dependent bindingability to hsIL-6R. In contrast, the disappearance of hsIL-6R wassignificantly accelerated when hsIL-6R was administered simultaneouslywith 6RL#9-IgG1 or FH4-IgG1 having 100-fold or higher Ca-dependentbinding ability to hsIL-6R. The plasma concentrations of hsIL-6R one dayafter hsIL-6R was administered simultaneously with 6RL#9-IgG1 andFH4-IgG1 were reduced 39-fold and 2-fold, respectively, as compared withsimultaneous administration with H54/L28-IgG1. Thus, thecalcium-dependent binding antibodies were confirmed to be able toaccelerate antigen disappearance from the plasma.

[Reference Example 19] Trials to Improve the AntigenElimination-Accelerating Effect of Antibody with Ca-DependentAntigen-Binding (Preparation of Antibodies) (19-1) Regarding the Bindingof IgG Antibody to FcRn

IgG antibodies have longer plasma retention time as a result of FcRnbinding. The binding between IgG and FcRn is observed only under anacidic condition (pH 6.0). By contrast, the binding is almostundetectable under a neutral condition (pH 7.4). An IgG antibody istaken up into cells in a nonspecific manner. The antibody returns to thecell surface by binding to endosomal FcRn under the endosomal acidiccondition, and then dissociates from FcRn under the plasma neutralcondition. When the FcRn binding under the acidic condition is lost byintroducing mutations into the IgG Fc region, the antibody retentiontime in plasma is markedly impaired because the antibody no longerrecycles to the plasma from the endosome.

A reported method for improving the plasma retention of an IgG antibodyis to enhance the FcRn binding under acidic conditions. Amino acidmutations are introduced into its Fc region of an IgG antibody toimprove its FcRn binding under acidic conditions. This increases theefficiency of recycling of IgG antibody to the plasma from the endosome,resulting in improvement of the plasma retention of IgG antibody. Whenintroducing amino acid substitution, it is considered important not toincrease the binding to FcRn under neutral conditions. IgG antibodiesthat bind to FcRn under neutral conditions can return onto the cellsurface through binding to FcRn under the acidic condition of theendosome, but IgG antibodies do not dissociate from the FcRn in plasmaunder neutral conditions and are not recycled to the plasma, and thusplasma retention of IgG antibodies was thought to be inversely impaired.

For example, as described by Dall'Acqua et al. (J. Immunol. (2002) 169(9), 5171-5180), the plasma retention of IgG1 antibody that was allowedto bind to mouse FcRn under a neutral condition (pH 7.4) was exacerbatedas a result of introducing an amino acid substitution into a mouse. Inaddition, as described by Yeung et al. (J. Immunol. (2009) 182 (12),7663-7671), Datta-Mannan et al. (J. Biol. Chem. (2007) 282 (3),1709-1717), and Dall'Acqua et al. (J. Immunol. (2002) 169 (9),5171-5180), IgG1 antibody variants whose binding to human FcRn under anacidic condition (pH 6.0) is improved by introducing an amino acidsubstitution is also observed to bind to human FcRn under a neutralcondition (pH 7.4). Reportedly, the plasma retention of said antibodyadministered to a cynomolgus monkey was not improved, showing no changein the plasma retention. Thus, in antibody engineering technology forimproving antibody functions, efforts have been made to improve theplasma retention of antibody by increasing its binding to human FcRnunder acidic conditions without increasing its binding to human FcRnunder a neutral condition (pH 7.4). In other words, no report has beenpublished on the advantages of IgG1 antibodies whose binding to humanFcRn under a neutral condition (pH 7.4) is increased by introducingamino acid substitutions into the Fc region.

Antibodies that bind to an antigen in a Ca-dependent manner areextremely useful, because they have an effect of accelerating thedisappearance of soluble antigen and the repeated binding of a singleantibody molecule to soluble antigen. A method of enhancing binding toFcRn under a neutral condition (pH 7.4) was examined as a method tofurther improve the accelerating effect on antigen disappearance.

(19-2) Preparation of Ca-Dependent Human IL-6 Receptor-BindingAntibodies Having FcRn-Binding Ability Under Neutral Conditions

An amino acid mutation was introduced into the Fc regions of FH4-IgG1and 6RL#9-IgG1 having a calcium-dependent antigen-binding ability andH54/L28-IgG1 having no calcium-dependent antigen-binding ability (usedas a control) to prepare variants having an FcRn-binding ability under aneutral condition (pH 7.4). The amino acid mutation was introduced by amethod known in the art using PCR. Specifically, FH4-N434W (heavy chainSEQ ID NO: 115, light chain SEQ ID NO: 101), 6RL#9-N434W (heavy chainSEQ ID NO: 116, light chain SEQ ID NO: 99), and H54/L28-N434W (heavychain SEQ ID NO: 117, light chain SEQ ID NO: 39) with Asn (an amino acidat position 434 represented by the EU numbering) substituted by Trp inthe heavy chain constant region of IgG1 were prepared. An animal cellexpression vector into which a polynucleotide encoding a variant withthe amino acid substitution was inserted was prepared using theQuikChange Site-Directed Mutagenesis Kit (Stratagene) by the methoddescribed in the accompanying instructions. Antibody expression andpurification, and concentration measurement were conducted according tothe method described in Reference Example 11.

[Reference Example 20] Examination of the Effect of AcceleratingDisappearance of Ca-Dependent Binding Antibodies Using Normal Mice(20-1) In Vivo Test Using Normal Mice

To a normal mouse (C57BL/6J mouse, Charles River Japan), hsIL-6R(soluble human IL-6 receptor prepared in Reference Example 20) alone wasadministered, or hsIL-6R and anti-human IL-6 receptor antibody wereadministered simultaneously to examine the kinetics of the hsIL-6R andanti-human IL-6 receptor antibody in vivo. A single dose (10 mL/kg) ofhsIL-6R solution (5 μg/mL) or a mixture of hsIL-6R and anti-human IL-6receptor antibody was administered into the caudal vein. The aboveH54/L28-N434W, 6RL#9-N434W, and FH4-N434W were used as anti-human IL-6receptor antibodies.

The concentration of hsIL-6R in all the mixtures is 5 μg/mL. Theconcentrations of anti-human IL-6 receptor antibody vary with theantibodies: prepared at 0.042 mg/mL for H54/L28-N434W, 0.55 mg/mL for6RL#9-N434W, and 1 mg/mL for FH4-N434W. At this time, it was thoughtthat most of the hsIL-6Rs bind to the antibody because the anti-humanIL-6 receptor antibody against hsIL-6R exists in a sufficient orexcessive amount. Blood samples were collected at 15 minutes, 7 hoursand 1, 2, 4, 7, 14, 21, and 28 days after the administration. The bloodsamples were immediately centrifuged for 15 minutes at 4° C. and 12,000rpm to separate plasma. The separated plasma was stored in a freezer setto −20° C. or lower until the time of measurement.

(20-2) Determination of Plasma Anti-Human IL-6 Receptor AntibodyConcentration in Normal Mice by ELISA

The plasma concentration of anti-human IL-6 receptor antibody in a mousewas determined by ELISA as described in Reference Example 18. Changes inthe plasma concentrations of antibodies, H54/L28-N434W, 6RL#9-N434W, andFH4-N434W, in the normal mice after intravenous administration measuredas described above are shown in FIG. 21.

(20-3) Determination of Plasma hsIL-6R Concentration by anElectrochemiluminescence Method

The plasma concentration of hsIL-6R in a mouse was determined by anelectrochemiluminescence method. An hsIL-6R calibration curve sampleprepared at 2,000, 1,000, 500, 250, 125, 62.5, or 31.25 pg/mL, and amouse plasma measurement sample diluted by 50-fold or above, were mixedwith a monoclonal anti-human IL-6R antibody (R&D) ruthenated withSULFO-TAG NHS Ester (Meso Scale Discovery) and a biotinylated anti-humanIL-6 R antibody (R&D), followed by overnight reaction at 4° C. At thattime, the assay buffer contained 10 mM EDTA to reduce the free Caconcentration in the sample and dissociate almost all hsIL-6Rs in thesample from 6RL#9-N434W or FH4-N434W to exist in a free state.Subsequently, said reaction liquid was dispensed into an MA400 PRStreptavidin Plate (Meso Scale Discovery). In addition, after washingeach well of the plate that was allowed to react for 1 hour at 25° C.,Read Buffer T (×4) (Meso Scale Discovery) was dispensed into each well.Immediately, the reaction liquid was subjected to measurement using aSECTOR PR 400 reader (Meso Scale Discovery). The concentration ofhsIL-6R was calculated from the response of the calibration curve usingthe SOFTmax PRO analysis software (Molecular Devices). Changes in theplasma concentration of hsIL-6R in the normal mouse after intravenousadministration determined as described above are shown in FIG. 22.

As a result, in comparison with the administration of hsIL-6R alone,simultaneous administration of hsIL-6R with the H54/L28-N434W antibodywhich has FcRn-binding activity at pH 7.4 and does not have Ca-dependentbinding activity to hsIL-6R had a significantly delayed disappearance ofhsIL-6R. In contrast, the disappearance of hsIL-6R was accelerated whenhsIL-6R was administered simultaneously with the 6RL#9-N434W orFH4-N434W antibody which has 100-fold or higher Ca-dependent bindingability to hsIL-6R and FcRn-binding activity at pH 7.4, as compared withthe administration of hsIL-6R alone. The plasma concentrations ofhsIL-6R one day after hsIL-6R was administered simultaneously with the6RL#9-N434W or FH4-N434W antibody were reduced 3-fold and 8-fold,respectively, as compared with the administration of hsIL-6R alone. As aresult, it was confirmed that the disappearance of antigen from plasmacould be further accelerated by imparting FcRn-binding activity at pH7.4 to an antibody that binds to antigen in a calcium-dependent manner.

The 6RL#9-IgG1 or FH4-IgG1 antibody having 100-fold or higherCa-dependent binding activity to hsIL-6R was confirmed to increase thedisappearance of hsIL-6R, as compared with the H54/L28-IgG1 antibodyhaving no Ca-dependent binding activity to hsIL-6R. The 6RL#9-N434W orFH4-N434W antibody which has 100-fold or higher Ca-dependent bindingactivity to hsIL-6R and FcRn-binding activity at pH 7.4 was confirmed tomore strongly accelerate the disappearance of hsIL-6R, as compared withthe administration of hsIL-6R alone.

These data suggest that an antibody that binds to an antigen in aCa-dependent manner dissociates from antigen in the endosome, similarlyto an antibody that binds to antigen in a pH-dependent manner.

(Reference Example 21) Preparation of Soluble Human IL-6 Receptor(hsIL-6R)

Recombinant human IL-6 receptor of the human IL-6 receptor, which is theantigen, was prepared as follows. A CHO cell line constitutivelyexpressing soluble human IL-6 receptor (hereinafter referred to ashsIL-6R) having the amino acid sequence of positions 1 to 357 from the Nterminus as reported by Mullberg et al. (J. Immunol. (1994) 152,4958-4968) was established by a method known to those skilled in theart. This expression line was cultured to express hsIL-6R. hsIL-6R waspurified from the obtained culture supernatant by Blue Sepharose 6 FFcolumn chromatography and gel filtration column chromatography. Afraction eluted as the main peak in the final step was used as the finalpurification product.

[Reference Example 22] Design of pH-Dependent Binding Antibody Library

(22-1) Method for Acquiring pH-Dependent Binding Antibodies

WO2009/125825 discloses a pH-dependent antigen-binding antibody whoseproperties are changed in neutral and acidic pH regions by introducing ahistidine into an antigen-binding molecule. The disclosed pH-dependentbinding antibody is obtained by modification to substitute a part of theamino acid sequence of the antigen-binding molecule of interest with ahistidine. To obtain a pH-dependent binding antibody more efficientlywithout preliminarily obtaining the antigen-binding molecule of interestto be modified, one method may be obtaining an antigen-binding moleculethat binds to a desired antigen from a population of antigen-bindingmolecules (referred to as His library) with a histidine introduced intothe variable region (more preferably, a region potentially involved inantigen binding). It may be possible to efficiently obtain anantigen-binding molecule having desired properties from a His library,because histidine appears more frequently in antigen-binding moleculesfrom His library than those from conventional antibody libraries.

(22-2) Design of a Population of Antibody Molecules (his Library) withHistidine Residue Introduced into their Variable Region to EffectivelyAcquire Binding Antibodies that Bind to Antigen in a pH-Dependent Manner

First, positions for introducing a histidine were selected in a Hislibrary. WO2009/125825 discloses generation of pH-dependentantigen-binding antibodies by substituting amino acid residues in thesequences of IL-6 receptor, IL-6, and IL-31 receptor antibodies with ahistidine. In addition, anti-egg white lysozyme (FEBS Letter 11483, 309,1, 85-88) and anti-hepcidin (WO 2009/139822) antibodies having apH-dependent antigen-binding ability were generated by substituting theamino acid sequence of the antigen-binding molecule with histidines.Positions where histidines were introduced in the IL-6 receptorantibody, IL-6 antibody, IL-31 receptor antibody, egg white lysozymeantibody, and hepcidin antibody are shown in Table 28. Positions shownin Table 28 may be listed as candidate positions that can control theantigen-antibody binding. In addition, besides the position shown inTable 28, positions that are likely to have contact with antigen werealso considered to be suitable for introduction of histidines.

TABLE 28 ANTIBODY CHAIN POSITION (Kabat) IL-6 RECEPTOR H 27 31 32 35 5058 62 100B 102 ANTIBODY L 28 31 32 53 56 92 IL-6 ANTIBODY H 32 59 61 99L 53 54 90 94 IL-31 RECEPTOR H 33 ANTIBODY L EGG-WHILE LYSOZYME H 33 98ANTIBODY L 54 HEPCIDIN ANTIBODY H 52 57 99 107 L 27 89

In the His library consisting of heavy-chain and light-chain variableregions, a human antibody sequence was used for the heavy chain variableregion, and histidines were introduced into the light chain variableregion. The positions listed above and positions that may be involved inantigen binding, i.e., positions 30, 32, 50, 53, 91, 92, and 93 (Kabatnumbering, Kabat E A et al. 1991. Sequence of Proteins of ImmunologicalInterest. NIH) in the light chain were selected as positions forintroducing histidines in the His library. In addition, the Vk1 sequencewas selected as a template sequence of the light chain variable regionfor introducing histidines. Multiple amino acids were allowed to appearin the template sequence to diversify antigen-binding molecules thatconstitute the library. Positions exposed on the surface of a variableregion that is likely to interact with the antigen were selected asthose where multiple amino acids are allowed to appear. Specifically,positions 30, 31, 32, 34, 50, 53, 91, 92, 93, 94, and 96 of the lightchain (Kabat numbering, Kabat E A et al. 1991. Sequence of Proteins ofImmunological Interest. NIH) were selected as flexible residues.

The type and appearance frequency of amino acid residues that weresubsequently allowed to appear were determined. The appearance frequencyof amino acids in the flexible residues in the hVk1 and hVk3 sequencesregistered in the Kabat database (KABAT, E. A. ET AL.: ‘Sequences ofproteins of immunological interest’, vol. 91, 1991, NIH PUBLICATION) wasanalyzed. Based on the analysis results, the type of amino acids thatwere allowed to appear in the His library were selected from those withhigher appearance frequency at each position. At this time, amino acidswhose appearance frequency was determined to be low based on theanalysis results were also selected to avoid the bias of amino acidproperties. The appearance frequency of the selected amino acids wasdetermined in reference to the analysis results of the Kabat database.

As His libraries, His library 1 which is fixed to necessarilyincorporate a single histidine into each CDR, and His library 2 which ismore emphasized on sequence diversity than the His library 1 weredesigned by taking the amino acids and appearance frequency set asdescribed above into consideration. The detailed designs of Hislibraries 1 and 2 are shown in Tables 3 and 4 (with the positions ineach table representing the Kabat numbering). Ser (S) at position 94 canbe excluded if position 92 represented by the Kabat numbering is Asn (N)for the appearance frequency of amino acids as described in Tables 3 and4.

[Reference Example 23] Preparation of a Phage Display Library for HumanAntibodies (his Library 1) to Obtain an Antibody that Binds to Antigenin a pH-Dependent Manner

A gene library of antibody heavy-chain variable regions was amplified byPCR using a poly A RNA prepared from human PBMC, and commercial humanpoly A RNA as a template. A gene library of antibody light-chainvariable regions designed as His library 1 as described in ReferenceExample 2 was amplified using PCR. A combination of the gene librariesof antibody heavy-chain and light-chain variable regions generated asdescribed above was inserted into a phagemid vector to construct a humanantibody phage display library which presents Fab domains consisting ofhuman antibody sequences. For the construction method, Methods Mol Biol.(2002) 178, 87-100 was used as a reference. For the construction of thelibrary, a linker region connecting the phagemid Fab to the phage pIIIprotein, and the sequences of a phage display library with a trypsincleavage sequence inserted between the N2 and CT domains of the helperphage pIII protein gene were used. Sequences of the antibody geneportions isolated from E. coli into which the antibody gene library wasintroduced were identified, and sequence information was obtained for132 clones. The designed amino acid distribution and the amino aciddistribution of the identified sequences are shown in FIG. 23. A librarycontaining various sequences corresponding to the designed amino aciddistribution was constructed.

[Reference Example 24] Isolation of Antibodies that Bind to IL-6R in apH-Dependent Manner (24-1) Isolation of Antibody Fragments, which Bindto Antigens in a pH-Dependent Manner, from the Library by Bead Panning

The first selection from the constructed His library 1 was performed byenriching only antibody fragments with antigen (IL-6R) binding ability.

Phages were produced by E. coli containing the constructed phagemids forphage display. To precipitate the phages, 2.5 M NaCl/10% PEG was addedto the E. coli culture fluid of phage production. The precipitated phagepopulation was diluted with TBS to prepare a phage library solution. BSAand CaCl₂ were added to the phage library solution to adjust the finalBSA concentration to 4% and the final calcium ion concentration to 1.2mM. Regarding the panning method, the present inventors referred togeneral panning methods using antigens immobilized onto magnetic beads(J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001)247 (1-2), 191-203; Biotechnol. Prog. (2002) 18(2) 212-20, Mol. CellProteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidincoated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidincoated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to theprepared phage library solution to allow the contact of the phagelibrary solution with the antigen at room temperature for 60 minutes.BSA-blocked magnetic beads were added and allowed to bind toantigen/phage complexes at room temperature for 15 minutes. The beadswere washed three times with 1 ml of 1.2 mM CaCl₂/TBST (TBS containing1.2 mM CaCl₂ and 0.1% Tween20) and then twice with 1 ml of 1.2 mMCaCl₂/TBS (pH 7.6). Then, the beads added with 0.5 ml of 1 mg/ml trypsinwere suspended at room temperature for 15 minutes, and then immediatelyseparated using a magnetic stand to collect a phage solution. Thecollected phage solution was added to 10 ml of E. coli strain ER2738 ina logarithmic growth phase (OD600 of 0.4-0.7). The E. coli was infectedwith the phages by culturing them while gently stirring at 37° C. forone hour. The infected E. coli was plated in a 225 mm×225 mm plate.Then, the phages were collected from the culture fluid of the plated E.coli to prepare a phage library solution.

To enrich the phages, the second and subsequent rounds of panning wereperformed using the antigen-binding ability or the pH-dependent bindingability as an indicator. Specifically, 40 pmol of the biotin-labeledantigen was added to the prepared phage library solution to allow thecontact of the phage library solution with the antigen at roomtemperature for 60 minutes. BSA-blocked magnetic beads were added andallowed to bind to antigen/phage complexes at room temperature for 15minutes. The beads were washed multiple times with 1 ml of 1.2 mMCaCl₂/TBST and with 1.2 mM CaCl₂/TBS. Then, when the phages wereenriched using the antigen-binding ability as an indicator, the beadsadded with 0.5 ml of 1 mg/ml trypsin were suspended at room temperaturefor 15 minutes, and then immediately separated using a magnetic stand tocollect a phage solution. Alternatively, when the phages were enrichedusing the pH-dependent antigen-binding ability as an indicator, thebeads added with 0.1 ml of 50 mM MES/1.2 mM CaCl_(2/150) mM NaCl (pH5.5) were suspended at room temperature, and then immediately separatedusing a magnetic stand to collect a phage solution. To eliminate theability from phages displaying no Fab to infect E. coli, the pIIIprotein (helper phage-derived pIII protein) of phages displaying no Fabwas cleaved by adding 5 μl of 100 mg/ml trypsin to the collected phagesolution. The collected phages were added to 10 ml of E. coli strainER2738 in a logarithmic growth phase (OD600 of 0.4-0.7). The E. coli wasinfected with the phages by culturing them while gently stirring at 37°C. for one hour. The infected E. coli was plated in a 225 mm×225 mmplate. Then, the phages were collected from the culture fluid of theplated E. coli to collect a phage library solution. The panning usingthe antigen-binding ability or the pH-dependent binding ability as anindicator was repeated twice.

(24-2) Assessment by Phage ELISA

Phage-containing culture supernatants were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above.

To the phage-containing culture supernatants, BSA and CaCl₂ were addedat a final concentration of 4% BSA and at a final calcium ionconcentration of 1.2 mM. These phage-containing culture supernatantswere subjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μl of PBScontaining the biotin-labeled antigen. After washing each well of theplate with PBST (PBS containing 0.1% Tween20) to remove the antigen, thewells were blocked with 250 μl of 4% BSA/TBS for one hour or more. Afterremoving 4% BSA/TBS, the prepared culture supernatants were added toeach well. The antibodies presented on the phages were allowed to bindto the antigens on each well by incubating the plate at 37° C. for onehour. Following wash with 1.2 mM CaCl₂/TBST, 1.2 mM CaCl₂/TBS (pH 7.6)or 1.2 mM CaCl₂/TBS (pH 5.5) was added to each well. The plate wasincubated at 37° C. for 30 minutes. After washing with 1.2 mM CaCl₂/TBST(pH 7.6), HRP-coupled anti-M13 antibody (Amersham Pharmacia Biotech)diluted with TBS containing 4% BSA and 1.2 mM ionized calcium was addedto each well. The plate was incubated for one hour. After washing with1.2 mM CaCl₂/TBST, TMB single solution (ZYMED) was added to each well.The chromogenic reaction in the solution of each well was stopped byadding sulfuric acid, and then the absorbance at 450 nm was measured toassess the color development.

When enrichment was carried out using the antigen-binding ability as anindicator, phage ELISA following two rounds of panning showed that 17 of96 clones were ELISA positive in an antigen-specific manner. Thus,clones were analyzed after three rounds of panning. Meanwhile, whenenrichment was carried out using the pH-dependent antigen-bindingability as an indicator, phage ELISA following two rounds of panningshowed that 70 of 94 clones were positive in ELISA. Thus, clones wereanalyzed after two rounds of panning.

The base sequences of genes amplified with specific primers wereanalyzed for the clones subjected to phage ELISA. The results of phageELISA and sequence analysis are shown in Table 29 below.

TABLE 29 LIBRARY His LIBRARY 1 His LIBRARY 1 ENRICHMENT INDEX ANTIGEN-pH-DEPENDENT BINDING ANTIGEN- ABILITY BINDING ABILITY NUMBER OF PANNING3 2 NUMBER OF 80 94 EXAMINED CLONES ELISA-POSITIVE 76 70 TYPES OF ELISA-30 67 POSITIVE CLONE SEQUENCES TYPES OF pH-DEPENDENT 22 47 BINDING CLONESEQUENCES

By the same method, antibodies with pH-dependent antigen-binding abilitywere isolated from the naive human antibody phage display library. Whenenrichment was carried out using the antigen-binding ability as anindicator, 13 types of pH-dependent binding antibodies were isolatedfrom 88 clones tested. Meanwhile, when enrichment was carried out usingthe pH-dependent antigen-binding ability as an indicator, 27 types ofpH-dependent binding antibodies were isolated from 83 clones tested.

The result described above demonstrated that the variation of cloneswith pH-dependent antigen-binding ability isolated from the His library1 was larger as compared to the naive human antibody phage displaylibrary.

(24-3) Expression and Purification of Antibodies

Clones assumed to have pH-dependent antigen-binding ability based on theresult of phage ELISA were introduced into animal cell expressionplasmids. Antibodies were expressed using the method described below.Cells of human fetal kidney cell-derived FreeStyle 293-F line(Invitrogen) were suspended in FreeStyle 293 Expression Medium(Invitrogen), and plated at a cell density of 1.33×10⁶ cells/ml (3 ml)to each well of a 6-well plate. The prepared plasmids were introducedinto the cells by a lipofection method. The cells were cultured in a CO₂incubator (37° C., 8% CO₂, 90 rpm) for four days. By a method known tothose skilled in the art, antibodies were purified using rProtein ASepharose™ Fast Flow (Amersham Biosciences) from culture supernatantsobtained as described above. The absorbance of solutions of purifiedantibodies was measured at 280 nm using a spectrophotometer. Antibodyconcentrations were calculated from the measured values by using theabsorption coefficient determined by PACE method (Protein Science (1995)4, 2411-2423).

(24-4) Assessment of Isolated Antibodies for their pH-Dependent BindingAbility to Human IL-6 Receptor

Antibodies 6RpH#01 (heavy chain SEQ ID NO: 118; light chain SEQ ID NO:119), 6RpH#02 (heavy chain SEQ ID NO: 120; light chain SEQ ID NO: 121),and 6RpH#03 (heavy chain SEQ ID NO: 122; light chain SEQ ID NO: 123)isolated as described in (24-3) were assessed for the pH dependency oftheir human IL-6 receptor-binding activity by analyzing the interactionbetween the antibodies and human IL-6 receptor using Biacore T100 (GEHealthcare). Tocilizumab (heavy chain SEQ ID NO: 60; light chain SEQ IDNO: 61) was used as a control antibody that does not have pH-dependentbinding activity to human IL-6 receptor. The interaction for theantigen-antibody reaction was analyzed in solutions at pH 7.4 and pH6.0, corresponding to a neutral pH and acidic pH conditions,respectively. An appropriate amount of Protein A/G (Invitrogen) wasimmobilized onto a Sensor chip CM5 (GE Healthcare) by an amino couplingmethod, and about 300 RU each of antibodies of interest were capturedonto the chip. The two types of running buffers used were: 20 mMACES/150 mM NaCl/0.05% (w/v) Tween20/1.2 mM CaCl₂ (pH 7.4); and 20 mMACES/150 mM NaCl/0.05% (w/v) Tween20/1.2 mM CaCl₂ (pH 6.0). Thesebuffers were each used to dilute human IL-6 receptor. All measurementswere carried out at 37° C.

In the interaction analysis of the antigen-antibody reaction usingtocilizumab as a control antibody, and antibodies 6RpH#01, 6RpH#02, and6RpH#03, a diluted IL-6 receptor solution and a running buffer as ablank were injected at a flow rate of 5 μl/min for three minutes toallow IL-6 receptor to interact with antibodies tocilizumab, 6RpH#01,6RpH#02, and 6RpH#03 captured onto the sensor chip. Then, 10 mMglycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/min for 30seconds to regenerate the sensor chip.

Sensorgrams at pH 7.4 obtained by the measurement using the methoddescribed above are shown in FIG. 24. Sensorgrams under the condition ofpH 6.0 obtained by the same method are shown in FIG. 25.

The result described above shows that the IL-6 receptor-binding abilityof antibodies 6RpH#01, 6RpH#02, and 6RpH#03 was significantly reducedwhen the buffer pH was shifted from pH 7.4 to pH 6.0.

(Reference Example 25) Production of Human IL-6 Receptor Knock-In Mice(25-1) Construction of a Knock-In Vector

A bacterial artificial chromosome (BAC) clone into which a genomicregion of mouse interleukin-6 gene (Il6ra) has been cloned was used. ADNA fragment in which the human interleukin-6 receptor gene codingsequence (GenBank # NM_000565_), hp7 sequence, poly A addition signal,loxP sequence, neomycin-resistance (neo) gene cassette, and loxP areserially linked was inserted into the target region of the mouse Il6ragene on this BAC by homologous recombination using a Red/ET system(GeneBridges). In this case, insertion was carried out by matching thetranslation initiation site located in exon 1 of the mouse Il6ra gene onBAC with the translation initiation site of the human IL6R gene, andonly 40 base pairs of the nucleotide sequence following the translationinitiation site in exon 1 of the mouse Il6ra gene were deleted. A pkggene promoter was added to neo, which is the drug-resistance gene, sothat the neo gene is expressed in ES cells. However, the neo gene ispredicted to possibly suppress the expression of the hIL6R geneintroduced into the upstream region. Therefore, to enable later removalof the neo gene, the loxP sequence (ATAACTTCGTATAGCATACATTATACGAAGTTAT(SEQ ID NO: 131)) was placed on both sides of the neo gene. Thisproduced a system where the neo gene situated between the loxP sequenceswill be removed by recombination when Cre acts on it. Next, to enablelinearization of the knock-in vector, the restriction enzyme NotIrecognition sequence (GCGGCCGC) was inserted together with theampicillin resistance gene to the 5′ upstream region of the mouse Il6ragene on BAC.

(25-2) Introduction into ES Cells

The above-mentioned hIL6R knock-in vector was electroporated into EScells (129SvEv mouse-derived cells), and after selective culturing withG418, drug-resistant clones were obtained. From these clones, homologousrecombinants were screened by the PCR method. 60 μg of the knock-invector was linearized with NotI, extracted with phenol/chloroform,precipitated with ethanol, and then dissolved in PBS.

To prepare PCR samples, ES cells to be used in the screening werecultured on a 96-well plate, washed twice using 200 μL of PBS per well,and then a cell lysis buffer having the following composition (5 μL of10× LA buffer II (TAKARA LA for Taq), 5 μL of 5% NP-40, 4 μL ofproteinase K (TAKARA) (20 mg/mL), and 36 μL of distilled water) wasadded thereto for treatment at 55° C. for two hours, followed bytreatment at 95° C. for 15 minutes to inactivate proteinase K.

A total volume of 25 μL of a PCR reaction mixture was prepared by mixing1 μL of the sample, 2.5 μL of 10× LA buffer II, 2.5 μL of 25 mM MgCl₂, 4μL of dNTP (including 2.5 mM each of dATP, dCTP, dGTP, and dTTP), 0.2 μLeach of the primers (50 μM each), 0.25 μL of LA Taq (TAKARA), and 14.35μL of distilled water. The PCR conditions were: preheating at 94° C. forfive minutes, 35 cycles of amplification consisting of 98° C. for tenseconds and 68° C. for 3 minutes 30 seconds, as well as heating at 68°C. for seven minutes.

P6Ra1 (forward) 5′-ACAGGGCCTTAGACTCACAGC-3′ (SEQ ID NO: 132) and hRLI611638R (reverse) 5′-AACTTGCTCCCGACACTACTGG-3′ (SEQ ID NO: 133) were usedfor the primers. Primer P6Ra1 was placed in the mouse Il6ra genomeregion at the 5′-side further upstream of the homologous arm of theknock-in vector, and hRLI6 11638R was placed inside the hIL6R cDNA (seeFIG. 32). In samples of ES cells that underwent homologousrecombination, an approximately 2.2-kb band is amplified.

(3) Generation of Knock-In Mice

The homologous recombinant ES clones were suspended by trypsintreatment, and washed with the ES cell medium. Five IU of equinechorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) wereadministered intraperitoneally at 48-hour intervals to femaleC57BL/6J(B6) mice to perform superovulation treatment, and these femalemice were crossed with male mice of the same lineage. The day when aplug was confirmed in a female mouse was regarded as day 0.5. Ongestation day 2.5, the uterus and the oviduct were perfused, and embryosin the 8-cell stage to the morula stage were collected. The collectedembryos were incubated overnight at 37° C., the embryos that developedinto blastocysts were used as host embryos and 10 to 15 ES cells wereinjected therein. The injected embryos were transplanted into the uterusof pseudopregnant ICR-type recipient females on gestation day 2.5, andthe offsprings were obtained 17 days later. Based on distinction by thecoat color of the offspring obtained by injection of ES cells to theblastocysts, chimeric mice with mixed presence of the recombinant EScells (wild-type color) and the host blastocyst-derived cells (black)were obtained. After maturation, the male chimeric mice were crossedwith the B6-female mice, and transmission of the knock-in allele to thenext generation mice was confirmed by the PCR method using the genomicDNA extracted from the tail of the next generation mice as the template.PCR was performed by the method used when screening for theabove-mentioned homologous recombinant ES cells. As a result,individuals from which a 2.2-kb signal was detected were obtained, andthe knock-in allele was confirmed to be transmitted to theseindividuals.

(4) Removal of the Neo Gene

The neo gene cassette was removed by microinjection of the recombinaseCre expression vector into the pronucleus of the fertilized egg obtainedby propagation of individuals in which transmission of the knock-inallele was confirmed. That is, by transiently expressing Cre,recombination was induced between the two loxP sites placed in theknock-in allele, and the neo gene cassette was removed. The fertilizedegg to which the Cre expression vector was microinjected was transferredto the oviducts of pseudopregnant ICR recipient females on gestation day0.5 and the offsprings were born 19 days later. Removal of the neo genecassette was confirmed by a PCR method using genomic DNA extracted fromthe tail collected after weaning of the offsprings.

The PCR reaction solution was composed of 1 μL of the sample, 12.5 μL of2× GC buffer I, 4 μL of dNTP (including 2.5 mM each of dATP, dCTP, dGTP,and dTTP), 0.25 μL each of the primers (50 μM each), 0.25 μL of LA Taq(TAKARA), and 6.75 μL of distilled water, and upon mixing them, thetotal amount was set to 25 μL. The PCR conditions were: preheating at94° C. for four minutes, 35 cycles of amplification consisting of 94° C.for 30 seconds, 62° C. for 30 seconds, and 72° C. for three minutes, aswell as heating at 72° C. for seven minutes.

The positions where the primers were set are shown in FIG. 32B. mRLI610355 (5′-TCTGCAGTAGCCTTCAAAGAGC-3′ (SEQ ID NO: 134)) and mRLI6 11166R(5′-AACCAGACAGTGTCACATTCC-3′ (SEQ ID NO: 135)) were used for theprimers. In the samples from individuals before removal of the neocassette, a 4.2-kb signal detected as an amplification product derivedfrom the knock-in allele and a 0.8-kb signal derived from the wild-typeallele were detected by PCR reaction, whereas in the samples fromindividuals whose neo cassette has been removed, an approximately 2.7-kbsignal and a 0.8-kb signal derived from the wild-type allele weredetected (FIG. 33).

(6) Confirmation of Human IL6R Expression and Mouse IL6ra Expression(6-1) Confirmation by the RT-PCR Method Using Tissue RNA

Analyses of expression of human IL6R and mouse IL6ra were carried out bythe RT-PCR method using tissue RNA of homozygous knock-in mice andwild-type mice. Tissue RNAs were prepared from the liver, spleen,thymus, kidney, heart and lung. Using 1 μg each of the tissue RNAs astemplates, cDNAs were synthesized by performing a reverse transcriptionreaction using a SuperScript II First Strand cDNA Synthesis Kit(Invitrogen) and oligo dT (20) primer. Human IL6R and mouse IL6ra weredetected by performing PCR using the synthesized cDNAs as templates.Human IL6R was detected using the combination of a forward primer6RIK-s1 (5′-CCCGGCTGCGGAGCCGCTCTGC-3′ (SEQ ID NO: 136)) set in the 5′untranslated region further upstream of the translation initiation sitewhich is the insertion position of the hIL6R gene in the knock-in alleleand a human IL6R-specific reverse primer RLI6-a1(5′-ACAGTGATGCTGGAGGTCCTT-3′ (SEQ ID NO: 137)). On the other hand, mouseIL6ra was detected by using the combination of the above-mentionedforward primer 6RIK-s1 and reverse primer 6RLIcA2(5′-AGCAACACCGTGAACTCCTTTG-3′ (SEQ ID NO: 138)) which is specific tomouse IL6ra. The PCR reaction solution was composed of 12.5 μL of thesample, 12.5 μL of 2× GC buffer I, 4 μL of dNTP (including 2.5 mM eachof dATP, dCTP, dGTP, and dTTP), 0.25 μL each of the primers (50 μMeach), 0.25 μL of LA Taq (TAKARA), and 6.75 μL of distilled water, andupon mixing them, the total amount was set to 25 μL. The PCR conditionswere: preheating at 94° C. for two minutes, 30 cycles of amplificationconsisting of 94° C. for 30 seconds, 62° C. for 30 seconds, and 72° C.for one minute, as well as heating at 72° C. for five minutes. While theamplification product of human IL6R is detected at 880 bp and theamplification product of mouse IL6ra is detected at 846 bp, only humanIL6R was detected from each of the tissues of homozygous hIL6R knock-inmice, and mouse IL6ra was not detected. Meanwhile, human IL6R was notdetected from each of the tissues of wild-type mice, and only mouseIL6ra was detected (FIG. 34). These results confirmed that the knock-invector underwent homologous recombination as designed, giving mice thatexpress human IL6R instead of mouse IL6ra.

(6-2) Measurement of Human IL-6R Concentration in the Plasma

Laparotomy was performed under isoflurane inhalation anesthesia, and theconcentration of soluble human IL-6R in the plasma separated from theblood collected from the abdominal large vein was measured using aQuantikine Human IL-6sR Immunoassay Kit (R&D Systems). As a result, theconcentration of soluble hIL-6R in plasma was 22.1±5.0 ng/mL for thehomozygous knock-in mice, and 11.5±4.1 ng/mL for the heterozygousknock-in mice. Soluble hIL-6R was not detected in the plasma ofwild-type mice (FIG. 35). The concentration in homozygous knock-in micewas equivalent to the concentration in blood reported for humans (Blood(2008) 112, 3959-3969).

(6-3) Confirmation of Species-Specific Ligand Reactivity

Mouse IL-6 or human IL-6 were administered intraperitoneally tohomozygous knock-in mice and wild-type mice at 4 μg per kg body weight,blood was collected six hours later, and the concentration of serumamyloid A (SAA) in the blood was quantified using an SAA ELISA Kit(Invitrogen). A solution produced by supplementing phosphate bufferedsaline solution (PBS) with mouse plasma so that it will become 0.5% wasused as the solvent of IL-6 for administration. A control group to whichonly the solvent is administered was prepared. As a result, thehomozygous knock-in mouse was responsive to human IL-6 only and theplasma SAA level increased, but it did not show any responsiveness tomouse IL-6 (FIG. 36). On the other hand, the wild-type mice wereresponsive to human IL-6 and mouse IL-6, and showed an increase in theplasma SAA level (FIG. 36). It is known that while mouse IL-6ra binds tomouse IL-6 as well as human IL-6, human IL-6R binds to human IL-6 butdoes not bind to mouse IL-6, and the results of this experiment were inaccordance with this knowledge. Therefore, it was revealed that in thehomozygous knock-in mice, mouse IL6ra is not expressed, and instead,human IL6R is expressed and is functioning, as designed.

Since the mRNA of the hIL6R gene transcribed by the knock-in allele ofthe present invention has a structure that will not be spliced out, itis not degraded by the NMD mechanism, but on the other hand, theexpression level of genes that are not spliced out is known to becomelow. However, in the hIL6R knock-in mice of the present invention, thesoluble hIL-6R concentration in the blood is the same as that in healthyindividuals, and moreover, the mice are sufficiently responsive to theadministered human IL-6 and SAA production was confirmed. This showsthat the hp7 inserted together with the poly A addition signalcontributed to the stabilization of the expression level of hIL6R whichwould normally have decreased because of its structure that is notspliced out.

1. A pharmaceutical composition that induces an immune response to anantigen, which comprises as an active ingredient an antigen-bindingmolecule, wherein the antigen-binding molecule comprises anantigen-binding domain whose binding activity to the antigen changesdepending on an ion concentration condition and comprises anFcRn-binding domain having binding activity to FcRn in a neutral pHrange.
 2. The pharmaceutical composition of claim 1, wherein the ionconcentration is a calcium ion concentration.
 3. The pharmaceuticalcomposition of claim 2, wherein the antigen-binding domain is anantigen-binding domain whose antigen-binding activity is higher under ahigh calcium ion concentration condition than under a low calcium ionconcentration condition.
 4. The pharmaceutical composition of claim 1,wherein the ion concentration condition is a pH condition.
 5. Thepharmaceutical composition of claim 4, wherein the antigen-bindingdomain is an antigen-binding domain whose antigen-binding activity ishigher in a neutral pH range than under an acidic pH range.
 6. Thepharmaceutical composition of any one of claims 1 to 5, wherein theantigen-binding molecule has neutralizing activity against the antigen.7. The pharmaceutical composition of any one of claims 1 to 6, whereinthe antigen-binding molecule has cytotoxic activity against a cellexpressing the antigen.
 8. The pharmaceutical composition of any one ofclaims 1 to 7, wherein the FcRn-binding domain comprises an antibody Fcregion.
 9. The pharmaceutical composition of claim 8, wherein the Fcregion is an Fc region in which at least one or more amino acidsselected from the group consisting of amino acids at positions 257, 308,428, and 434 according to EU numbering in the Fc region are differentfrom amino acids at corresponding positions in a naturally-occurring Fcregion.
 10. The pharmaceutical composition of claim 8 or 9, wherein theFc region comprises at least one or more amino acids selected from thegroup consisting of: Ala at amino acid position 257; Pro at amino acidposition 308; Leu at amino acid position 428; and Tyr at amino acidposition 434, according to EU numbering in the Fc region.
 11. Thepharmaceutical composition of any one of claims 8 to 10, wherein the Fcγreceptor-binding activity of the Fc region is higher than that of anaturally-occurring human IgG Fc region in which the sugar chainattached at position 297 according to EU numbering is afucose-containing sugar chain.
 12. The pharmaceutical composition ofclaim 11, wherein the Fcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H),FcγRIIb, FcγRIIIa(V), or FcγRIIIa(F).
 13. The pharmaceutical compositionof claim 11 or 12, wherein the Fc region is an Fc region in which atleast one or more amino acids selected from the group consisting ofamino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246,247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281,282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427,428, 429, 434, 436, and 440 according to EU numbering in the Fc regionare different from amino acids at corresponding positions in anaturally-occurring Fc region.
 14. The pharmaceutical composition of anyone of claims 11 to 13, wherein the Fc region comprises at least one ormore amino acids selected from the group consisting of: either Lys orTyr at amino acid position 221; any one of Phe, Trp, Glu, and Tyr atamino acid position 222; any one of Phe, Trp, Glu, and Lys at amino acidposition 223; any one of Phe, Trp, Glu, and Tyr at amino acid position224; any one of Glu, Lys, and Trp at amino acid position 225; any one ofGlu, Gly, Lys, and Tyr at amino acid position 227; any one of Glu, Gly,Lys, and Tyr at amino acid position 228; any one of Ala, Glu, Gly, andTyr at amino acid position 230; any one of Glu, Gly, Lys, Pro, and Tyrat amino acid position 231; any one of Glu, Gly, Lys, and Tyr at aminoacid position 232; any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position233; any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 234; anyone of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 235; any one of Ala,Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 236; any one of Asp, Glu, Phe,His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyrat amino acid position 237; any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acidposition 238; any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 239;any one of Ala, Ile, Met, and Thr at amino acid position 240; any one ofAsp, Glu, Leu, Arg, Trp, and Tyr at amino acid position 241; any one ofLeu, Glu, Leu, Gln, Arg, Trp, and Tyr at amino acid position 243; His atamino acid position 244; Ala at amino acid position 245; any one of Asp,Glu, His, and Tyr at amino acid position 246; any one of Ala, Phe, Gly,His, Ile, Leu, Met, Thr, Val, and Tyr at amino acid position 247; anyone of Glu, His, Gln, and Tyr at amino acid position 249; either Glu orGln at amino acid position 250; Phe at amino acid position 251; any oneof Phe, Met, and Tyr at amino acid position 254; any one of Glu, Leu,and Tyr at amino acid position 255; any one of Ala, Met, and Pro atamino acid position 256; any one of Asp, Glu, His, Ser, and Tyr at aminoacid position 258; any one of Asp, Glu, His, and Tyr at amino acidposition 260; any one of Ala, Glu, Phe, Ile, and Thr at amino acidposition 262; any one of Ala, Ile, Met, and Thr at amino acid position263; any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position 264; any one ofAla, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Val, Trp, and Tyr at amino acid position 265; any one of Ala,Ile, Met, and Thr at amino acid position 266; any one of Asp, Glu, Phe,His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr atamino acid position 267; any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu,Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268; anyone of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, and Tyr at amino acid position 269; any one of Glu, Phe, Gly, His,Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acidposition 270; any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position271; any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 272; either Phe or Ile atamino acid position 273; any one of Asp, Glu, Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position274; either Leu or Trp at amino acid position 275; any one of Asp, Glu,Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr atamino acid position 276; any one of Asp, Glu, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position278; Ala at amino acid position 279; any one of Ala, Gly, His, Lys, Leu,Pro, Gln, Trp, and Tyr at amino acid position 280; any one of Asp, Lys,Pro, and Tyr at amino acid position 281; any one of Glu, Gly, Lys, Pro,and Tyr at amino acid position 282; any one of Ala, Gly, His, Ile, Lys,Leu, Met, Pro, Arg, and Tyr at amino acid position 283; any one of Asp,Glu, Leu, Asn, Thr, and Tyr at amino acid position 284; any one of Asp,Glu, Lys, Gln, Trp, and Tyr at amino acid position 285; any one of Glu,Gly, Pro, and Tyr at amino acid position 286; any one of Asn, Asp, Glu,and Tyr at amino acid position 288; any one of Asp, Gly, His, Leu, Asn,Ser, Thr, Trp, and Tyr at amino acid position 290; any one of Asp, Glu,Gly, His, Ile, Gln, and Thr at amino acid position 291; any one of Ala,Asp, Glu, Pro, Thr, and Tyr at amino acid position 292; any one of Phe,Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr atamino acid position 293; any one of Phe, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 294;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 295; any one of Ala, Asp,Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val atamino acid position 296; any one of Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acidposition 297; any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn,Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 298; any one ofAla, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Val, Trp, and Tyr at amino acid position 299; any one of Ala, Asp,Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,and Trp at amino acid position 300; any one of Asp, Glu, His, and Tyr atamino acid position 301; Ile at amino acid position 302; any one of Asp,Gly, and Tyr at amino acid position 303; any one of Asp, His, Leu, Asn,and Thr at amino acid position 304; any one of Glu, Ile, Thr, and Tyr atamino acid position 305; any one of Ala, Asp, Asn, Thr, Val, and Tyr atamino acid position 311; Phe at amino acid position 313; Leu at aminoacid position 315; either Glu or Gln at amino acid position 317; any oneof His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at amino acidposition 318; any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser,Thr, Val, Trp, and Tyr at amino acid position 320; any one of Ala, Asp,Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr at amino acidposition 322; Ile at amino acid position 323; any one of Asp, Phe, Gly,His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at amino acidposition 324; any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position325; any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser,Thr, Val, Trp, and Tyr at amino acid position 326; any one of Ala, Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp,and Tyr at amino acid position 327; any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atamino acid position 328; any one of Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acidposition 329; any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 330;any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, andTyr at amino acid position 331; any one of Ala, Asp, Glu, Phe, Gly, His,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at aminoacid position 332; any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu,Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333; any one ofAla, Glu, Phe, Ile, Leu, Pro, and Thr at amino acid position 334; anyone of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp,and Tyr at amino acid position 335; any one of Glu, Lys, and Tyr atamino acid position 336; any one of Glu, His, and Asn at amino acidposition 337; any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg,Ser, and Thr at amino acid position 339; either Ala or Val at amino acidposition 376; either Gly or Lys at amino acid position 377; Asp at aminoacid position 378; Asn at amino acid position 379; any one of Ala, Asn,and Ser at amino acid position 380; either Ala or Ile at amino acidposition 382; Glu at amino acid position 385; Thr at amino acid position392; Leu at amino acid position 396; Lys at amino acid position 421; Asnat amino acid position 427; either Phe or Leu at amino acid position428; Met at amino acid position 429; Trp at amino acid position 434; Ileat amino acid position 436; and any one of Gly, His, Ile, Leu, and Tyrat amino acid position 440; according to EU numbering in the Fc region.15. The pharmaceutical composition of any one of claims 11 to 14,wherein the naturally-occurring Fc region is an Fc region of any one ofhuman IgG1, human IgG2, human IgG3, and human IgG4 in which the sugarchain attached at position 297 according to EU numbering is afucose-containing sugar chain.
 16. The pharmaceutical composition of anyone of claims 11 to 15, wherein the Fc region is modified so that thepercentage of the Fc region to which a fucose-deficient sugar chain isattached, or bisecting N-acetylglucosamine is added, at position 297according to EU numbering in the Fc region, will become higher.
 17. Amethod for inducing an immune response in a living organism, whichcomprises the step of administering the antigen-binding molecule of anyone of claims 1 to 16 to the living organism.
 18. A method for producingan antigen-binding molecule that induces an immune response, whichcomprises imparting FcRn-binding activity in a neutral pH range to anFcRn-binding domain that is contained in an antigen-binding moleculecontaining an antigen-binding domain whose antigen-binding activitychanges depending on an ion concentration condition.
 19. The method ofclaim 18, wherein the ion concentration is a calcium ion concentration.20. The method of claim 19, wherein the antigen-binding domain is anantigen-binding domain whose antigen-binding activity is higher under ahigh calcium ion concentration condition than under a low calcium ionconcentration condition.
 21. The method of claim 18, wherein the ionconcentration condition is a pH condition.
 22. The method of claim 21,wherein the antigen-binding domain is an antigen-binding domain whoseantigen-binding activity is higher in a neutral pH range than in anacidic pH range.
 23. The method of any one of claims 18 to 22, whereinthe antigen-binding molecule has neutralizing activity against theantigen.
 24. The method of any one of claims 18 to 23, wherein theantigen-binding molecule has cytotoxic activity against a cellexpressing the antigen.
 25. The method of any one of claims 18 to 24,wherein the FcRn-binding domain comprises an antibody Fc region.
 26. Themethod of claim 25, comprising the step of substituting at least one ormore amino acids selected from the group consisting of amino acids atpositions 239, 252, 257, 286, 307, 308, 428, and 434 according to EUnumbering in the Fc region.
 27. The method of claim 25 or 26, comprisingthe step of performing at least one or more amino acid substitutionsselected from the group consisting of: amino acid substitution with Alaat position 257; amino acid substitution with Pro at position 308; aminoacid substitution with Leu at position 428; and amino acid substitutionwith Tyr at position 434, according to EU numbering in the Fc region.28. The method of any one of claims 25 to 27, comprising the step ofenhancing the Fcγ receptor-binding activity of the Fc region as comparedto that of a naturally-occurring human IgG Fc region in which the sugarchain attached at position 297 according to EU numbering is afucose-containing sugar chain.
 29. The method of claim 28, wherein theFcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), orFcγRIIIa(F).
 30. The method of claim 28 or 29, comprising the step ofsubstituting at least one or more amino acids selected from the groupconsisting of amino acids at positions 221, 222, 223, 224, 225, 227,228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243,244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278,279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315,317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392,396, 421, 427, 428, 429, 434, 436, and 440 according to EU numbering inthe Fc region.
 31. The method of any one of claims 28 to 30, comprisingthe step of performing at least one or more amino acid substitutionsselected from the group consisting of: amino acid substitution witheither Lys or Tyr at position 221; amino acid substitution with any oneof Phe, Trp, Glu, and Tyr at position 222; amino acid substitution withany one of Phe, Trp, Glu, and Lys at position 223; amino acidsubstitution with any one of Phe, Trp, Glu, and Tyr at position 224;amino acid substitution with any one of Glu, Lys, and Trp at position225; amino acid substitution with any one of Glu, Gly, Lys, and Tyr atposition 227; amino acid substitution with any one of Glu, Gly, Lys, andTyr at position 228; amino acid substitution with any one of Ala, Glu,Gly, and Tyr at position 230; amino acid substitution with any one ofGlu, Gly, Lys, Pro, and Tyr at position 231; amino acid substitutionwith any one of Glu, Gly, Lys, and Tyr at position 232; amino acidsubstitution with any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 233; aminoacid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile,Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position234; amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 235; amino acid substitution with any one of Ala, Asp, Glu,Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp,and Tyr at position 236; amino acid substitution with any one of Asp,Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp, and Tyr at position 237; amino acid substitution with any one ofAsp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, and Tyr at position 238; amino acid substitution with any oneof Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val, Trp, and Tyr at position 239; amino acid substitution with any oneof Ala, Ile, Met, and Thr at position 240; amino acid substitution withany one of Asp, Glu, Leu, Arg, Trp, and Tyr at position 241; amino acidsubstitution with any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr atposition 243; amino acid substitution with His at position 244; aminoacid substitution with Ala at position 245; amino acid substitution withany one of Asp, Glu, His, and Tyr at position 246; amino acidsubstitution with any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr,Val, and Tyr at position 247; amino acid substitution with any one ofGlu, His, Gln, and Tyr at position 249; amino acid substitution witheither Glu or Gln at position 250; amino acid substitution with Phe atposition 251; amino acid substitution with any one of Phe, Met, and Tyrat position 254; amino acid substitution with any one of Glu, Leu, andTyr at position 255; amino acid substitution with any one of Ala, Met,and Pro at position 256; amino acid substitution with any one of Asp,Glu, His, Ser, and Tyr at position 258; amino acid substitution with anyone of Asp, Glu, His, and Tyr at position 260; amino acid substitutionwith any one of Ala, Glu, Phe, Ile, and Thr at position 262; amino acidsubstitution with any one of Ala, Ile, Met, and Thr at position 263;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position264; amino acid substitution with any one of Ala, Leu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyrat position 265; amino acid substitution with any one of Ala, Ile, Met,and Thr at position 266; amino acid substitution with any one of Asp,Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp,and Tyr at position 267; amino acid substitution with any one of Asp,Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp atposition 268; amino acid substitution with any one of Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr atposition 269; amino acid substitution with any one of Glu, Phe, Gly,His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at position270; amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 271; amino acid substitution with any one of Asp, Phe, Gly,His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr atposition 272; amino acid substitution with either Phe or Ile at position273; amino acid substitution with any one of Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position274; amino acid substitution with either Leu or Trp at position 275;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 276; aminoacid substitution with any one of Asp, Glu, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at position 278; aminoacid substitution with Ala at position 279; amino acid substitution withany one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at position280; amino acid substitution with any one of Asp, Lys, Pro, and Tyr atposition 281; amino acid substitution with any one of Glu, Gly, Lys,Pro, and Tyr at position 282; amino acid substitution with any one ofAla, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at position 283;amino acid substitution with at position 284 is any one of Asp, Glu,Leu, Asn, Thr, and Tyr; amino acid substitution with any one of Asp,Glu, Lys, Gln, Trp, and Tyr at position 285; amino acid substitutionwith any one of Glu, Gly, Pro, and Tyr at position 286; amino acidsubstitution with any one of Asn, Asp, Glu, and Tyr at position 288;amino acid substitution with any one of Asp, Gly, His, Leu, Asn, Ser,Thr, Trp, and Tyr at position 290; amino acid substitution with any oneof Asp, Glu, Gly, His, Ile, Gln, and Thr at position 291; amino acidsubstitution with any one of Ala, Asp, Glu, Pro, Thr, and Tyr atposition 292; amino acid substitution with any one of Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position293; amino acid substitution with any one of Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 294;amino acid substitution with any one of Asp, Glu, Phe, Gly, His, Ile,Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at position 295;amino acid substitution with any one of Ala, Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at position 296; aminoacid substitution with any one of Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 297;amino acid substitution with any one of Ala, Asp, Glu, Phe, His, Ile,Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at position 298; aminoacid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at position299; amino acid substitution with any one of Ala, Asp, Glu, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp atposition 300; amino acid substitution with any one of Asp, Glu, His, andTyr at position 301; amino acid substitution with Ile at position 302;amino acid substitution with any one of Asp, Gly, and Tyr at position303; amino acid substitution with at position 304 is any one of Asp,His, Leu, Asn, and Thr; amino acid substitution with any one of Glu,Ile, Thr, and Tyr at position 305; amino acid substitution with any oneof Ala, Asp, Asn, Thr, Val, and Tyr at position 311; amino acidsubstitution with Phe at position 313; amino acid substitution with Leuat position 315; amino acid substitution with either Glu or Gln atposition 317; amino acid substitution with any one of His, Leu, Asn,Pro, Gln, Arg, Thr, Val, and Tyr at position 318; amino acidsubstitution with any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro,Ser, Thr, Val, Trp, and Tyr at position 320; amino acid substitutionwith any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp,and Tyr at position 322; amino acid substitution with Ile at position323; amino acid substitution with any one of Asp, Phe, Gly, His, Ile,Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at position 324; amino acidsubstitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 325;amino acid substitution with any one of Ala, Asp, Glu, Gly, Ile, Leu,Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at position 326; aminoacid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at position 327;amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr atposition 328; amino acid substitution with any one of Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyrat position 329; amino acid substitution with any one of Cys, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyrat position 330; amino acid substitution with any one of Asp, Phe, His,Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr at position 331; aminoacid substitution with any one of Ala, Asp, Glu, Phe, Gly, His, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position332; amino acid substitution with any one of Ala, Asp, Glu, Phe, Gly,His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at position 333; aminoacid substitution with any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thrat position 334; amino acid substitution with any one of Asp, Phe, Gly,His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at position335; amino acid substitution with any one of Glu, Lys, and Tyr atposition 336; amino acid substitution with any one of Glu, His, and Asnat position 337; amino acid substitution with any one of Asp, Phe, Gly,Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr at position 339; amino acidsubstitution with either Ala or Val at position 376; amino acidsubstitution with either Gly or Lys at position 377; amino acidsubstitution with Asp at position 378; amino acid substitution with Asnat position 379; amino acid substitution with any one of Ala, Asn, andSer at position 380; amino acid substitution with either Ala or Ile atposition 382; amino acid substitution with Glu at position 385; aminoacid substitution with Thr at position 392; amino acid substitution withLeu at position 396; amino acid substitution with Lys at position 421;amino acid substitution with Asn at position 427; amino acidsubstitution with either Phe or Leu at position 428; amino acidsubstitution with Met at position 429; amino acid substitution with Trpat position 434; amino acid substitution with Ile at position 436; andamino acid substitution with any one of Gly, His, Ile, Leu, and Tyr atposition 440, according to EU numbering in the Fc region.
 32. The methodof any one of claims 28 to 31, wherein the naturally-occurring Fc regionis an Fc region of any one of human IgG1, human IgG2, human IgG3, andhuman IgG4 in which the sugar chain attached at position 297 accordingto EU numbering is a fucose-containing sugar chain.
 33. The method ofany one of claims 28 to 32, comprising the step of modifying the Fcregion so that the percentage of the Fc region to which afucose-deficient sugar chain is attached, or bisectingN-acetylglucosamine is added, at position 297 according to EU numberingin the Fc region, will become higher.
 34. A method for producing apharmaceutical composition which induces an immune response, whichcomprises the steps of: (a) determining the antigen-binding activity ofan antigen-binding domain under a high calcium ion concentrationcondition; (b) determining the antigen-binding activity of theantigen-binding domain under a low calcium ion concentration condition;(c) selecting the antigen-binding domain whose antigen-binding activitydetermined in (a) is higher than that determined in (b); (d) linking apolynucleotide encoding the antigen-binding domain selected in (c) to apolynucleotide encoding an FcRn-binding domain having FcRn-bindingactivity in a neutral pH range; (e) culturing a cell into which a vectorto which the polynucleotide obtained in (d) is operably linked has beenintroduced; and (f) collecting an antigen-binding molecule from culturefluid of the cell cultured in (e).
 35. A method for producing apharmaceutical composition which induces an immune response, whichcomprises the steps of: (a) determining the antigen-binding activity ofan antibody under a high calcium ion concentration condition; (b)determining the antigen-binding activity of the antibody under a lowcalcium ion concentration condition; (c) selecting the antibody whoseantigen-binding activity determined in (a) is higher than thatdetermined in (b); (d) linking a polynucleotide encoding theantigen-binding domain of the antibody selected in (c) to apolynucleotide encoding an FcRn-binding domain having FcRn-bindingactivity in a neutral pH range; (e) culturing a cell into which a vectorto which the polynucleotide obtained in (d) is operably linked has beenintroduced; and (f) collecting an antigen-binding molecule from culturefluid of the cell cultured in (e).
 36. A method for producing anantigen-binding molecule, which comprises the steps of: (a) determiningthe antigen-binding activity of an antigen-binding domain in a neutralpH range; (b) determining the antigen-binding activity of theantigen-binding domain in an acidic pH range; (c) selecting theantigen-binding domain whose antigen-binding activity determined in (a)is higher than that determined in (b); (d) linking a polynucleotideencoding the antigen-binding domain selected in (c) to a polynucleotideencoding an FcRn-binding domain having FcRn-binding activity in aneutral pH range; (e) culturing a cell into which a vector to which thepolynucleotide obtained in (d) is operably linked has been introduced;and (f) collecting an antigen-binding molecule from culture fluid of thecell cultured in (e).
 37. A method for producing an antigen-bindingmolecule, which comprises the steps of: (a) determining theantigen-binding activity of an antibody in a neutral pH range; (b)determining the antigen-binding activity of the antibody in an acidic pHrange; (c) selecting the antibody whose antigen-binding activitydetermined in (a) is higher than that determined in (b); (d) linking apolynucleotide encoding the antigen-binding domain of the antibodyselected in (c) to a polynucleotide encoding an FcRn-binding domainhaving FcRn-binding activity in a neutral pH range; (e) culturing a cellinto which a vector to which the polynucleotide obtained in (d) isoperably linked has been introduced; and (f) collecting anantigen-binding molecule from culture fluid of the cell cultured in (e).38. The method of any one of claims 34 to 37, wherein theantigen-binding molecule has neutralizing activity against the antigen.39. The method of any one of claims 34 to 38, wherein theantigen-binding molecule has cytotoxic activity against a cellexpressing the antigen.
 40. The method of any one of claims 34 to 39,wherein the FcRn-binding domain comprises an antibody Fc region.
 41. Themethod of claim 40, wherein the Fc region is an Fc region in which atleast one or more amino acids selected from the group consisting ofamino acids at positions 257, 308, 428, and 434 according to EUnumbering in the Fc region are different from amino acids atcorresponding positions in a naturally-occurring Fc region.
 42. Themethod of claim 40 or 41, wherein the Fc region comprises at least oneor more amino acids selected from the group consisting of: Ala at aminoacid position 257; Pro at amino acid position 308; Leu at amino acidposition 428; and Tyr at amino acid position 434, according to EUnumbering in the Fc region.
 43. The method of any one of claims 40 to42, wherein the Fcγ receptor-binding activity of the Fc region is higherthan that of a naturally-occurring human IgG Fc region in which thesugar chain attached at position 297 according to EU numbering is afucose-containing sugar chain.
 44. The method of claim 43, wherein theFcγ receptor is FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, FcγRIIIa(V), orFcγRIIIa(F).
 45. The method of claim 43 or 44, wherein the Fc regioncomprises at least one or more amino acids selected from the groupconsisting of: either Lys or Tyr at amino acid position 221; any one ofPhe, Trp, Glu, and Tyr at amino acid position 222; any one of Phe, Trp,Glu, and Lys at amino acid position 223; any one of Phe, Trp, Glu, andTyr at amino acid position 224; any one of Glu, Lys, and Trp at aminoacid position 225; any one of Glu, Gly, Lys, and Tyr at amino acidposition 227; any one of Glu, Gly, Lys, and Tyr at amino acid position228; any one of Ala, Glu, Gly, and Tyr at amino acid position 230; anyone of Glu, Gly, Lys, Pro, and Tyr at amino acid position 231; any oneof Glu, Gly, Lys, and Tyr at amino acid position 232; any one of Ala,Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp, and Tyr at amino acid position 233; any one of Ala, Asp, Glu, Phe,Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyrat amino acid position 234; any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at aminoacid position 235; any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acidposition 236; any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 237;any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 238; any one of Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val,Trp, and Tyr at amino acid position 239; any one of Ala, Ile, Met, andThr at amino acid position 240; any one of Asp, Glu, Leu, Arg, Trp, andTyr at amino acid position 241; any one of Leu, Glu, Leu, Gln, Arg, Trp,and Tyr at amino acid position 243; His at amino acid position 244; Alaat amino acid position 245; any one of Asp, Glu, His, and Tyr at aminoacid position 246; any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr,Val, and Tyr at amino acid position 247; any one of Glu, His, Gln, andTyr at amino acid position 249; either Glu or Gln at amino acid position250; Phe at amino acid position 251; any one of Phe, Met, and Tyr atamino acid position 254; any one of Glu, Leu, and Tyr at amino acidposition 255; any one of Ala, Met, and Pro at amino acid position 256;any one of Asp, Glu, His, Ser, and Tyr at amino acid position 258; anyone of Asp, Glu, His, and Tyr at amino acid position 260; any one ofAla, Glu, Phe, Ile, and Thr at amino acid position 262; any one of Ala,Ile, Met, and Thr at amino acid position 263; any one of Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyrat amino acid position 264; any one of Ala, Leu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Val, Trp, and Tyr atamino acid position 265; any one of Ala, Ile, Met, and Thr at amino acidposition 266; any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,Pro, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 267; anyone of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val,and Trp at amino acid position 268; any one of Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acidposition 269; any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln,Arg, Ser, Thr, Trp, and Tyr at amino acid position 270; any one of Ala,Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 271; any one of Asp, Phe, Gly,His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at aminoacid position 272; either Phe or Ile at amino acid position 273; any oneof Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, and Tyr at amino acid position 274; either Leu or Trp at amino acidposition 275; any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro,Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 276; any one ofAsp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, and Trp at amino acid position 278; Ala at amino acid position 279;any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at amino acidposition 280; any one of Asp, Lys, Pro, and Tyr at amino acid position281; any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 282;any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at aminoacid position 283; any one of Asp, Glu, Leu, Asn, Thr, and Tyr at aminoacid position 284; any one of Asp, Glu, Lys, Gln, Trp, and Tyr at aminoacid position 285; any one of Glu, Gly, Pro, and Tyr at amino acidposition 286; any one of Asn, Asp, Glu, and Tyr at amino acid position288; any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at aminoacid position 290; any one of Asp, Glu, Gly, His, Ile, Gln, and Thr atamino acid position 291; any one of Ala, Asp, Glu, Pro, Thr, and Tyr atamino acid position 292; any one of Phe, Gly, His, Ile, Leu, Met, Asn,Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 293; anyone of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, and Tyr at amino acid position 294; any one of Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at aminoacid position 295; any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296; anyone of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 297; any one of Ala, Asp,Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr atamino acid position 298; any one of Ala, Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at amino acidposition 299; any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp at amino acid position 300;any one of Asp, Glu, His, and Tyr at amino acid position 301; Ile atamino acid position 302; any one of Asp, Gly, and Tyr at amino acidposition 303; any one of Asp, His, Leu, Asn, and Thr at amino acidposition 304; any one of Glu, Ile, Thr, and Tyr at amino acid position305; any one of Ala, Asp, Asn, Thr, Val, and Tyr at amino acid position311; Phe at amino acid position 313; Leu at amino acid position 315;either Glu or Gln at amino acid position 317; any one of His, Leu, Asn,Pro, Gln, Arg, Thr, Val, and Tyr at amino acid position 318; any one ofAsp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr atamino acid position 320; any one of Ala, Asp, Phe, Gly, His, Ile, Pro,Ser, Thr, Val, Trp, and Tyr at amino acid position 322; Ile at aminoacid position 323; any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro,Arg, Thr, Val, Trp, and Tyr at amino acid position 324; any one of Ala,Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr,Val, Trp, and Tyr at amino acid position 325; any one of Ala, Asp, Glu,Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at aminoacid position 326; any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr at amino acid position327; any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 328; anyone of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Val, Trp, and Tyr at amino acid position 329; any one of Cys, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp,and Tyr at amino acid position 330; any one of Asp, Phe, His, Ile, Leu,Met, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 331; anyone of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, and Tyr at amino acid position 332; any one of Ala,Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr atamino acid position 333; any one of Ala, Glu, Phe, Ile, Leu, Pro, andThr at amino acid position 334; any one of Asp, Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at amino acid position 335;any one of Glu, Lys, and Tyr at amino acid position 336; any one of Glu,His, and Asn at amino acid position 337; any one of Asp, Phe, Gly, Ile,Lys, Met, Asn, Gln, Arg, Ser, and Thr at amino acid position 339; eitherAla or Val at amino acid position 376; either Gly or Lys at amino acidposition 377; Asp at amino acid position 378; Asn at amino acid position379; any one of Ala, Asn, and Ser at amino acid position 380; either Alaor Ile at amino acid position 382; Glu at amino acid position 385; Thrat amino acid position 392; Leu at amino acid position 396; Lys at aminoacid position 421; Asn at amino acid position 427; either Phe or Leu atamino acid position 428; Met at amino acid position 429; Trp at aminoacid position 434; Ile at amino acid position 436; and any one of Gly,His, Ile, Leu, and Tyr at amino acid position 440; according to EUnumbering in the Fc region.
 46. The method of any one of claims 43 to45, wherein the naturally-occurring Fc region is an Fc region of any oneof human IgG1, human IgG2, human IgG3, and human IgG4 in which the sugarchain attached at position 297 according to EU numbering is afucose-containing sugar chain.
 47. The method of any one of claims 43 to46, wherein the Fc region is modified so that the percentage of the Fcregion to which a fucose-deficient sugar chain is attached, or bisectingN-acetylglucosamine is added, at position 297 according to EU numberingin the Fc region, will become higher.